Engineered transaminase polypeptides

ABSTRACT

The present disclosure provides engineered transaminase polypeptides useful for the synthesis of chiral amine compounds under industrially relevant conditions. The disclosure also provides polynucleotides encoding the engineered transaminase polypeptides, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases for the production of chiral amine compounds.

The present application is a divisional application of U.S. patentapplication Ser. No. 17/223,677 filed Apr. 6, 2021 which claims thebenefit of U.S. Prov. Pat. Appln. Ser. No. 63/008,047, filed Apr. 10,2020, both of which are incorporated by reference in their entirety, forall purposes.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an XML file, with a filename of “CX2-197WO1_ST26”,a creation date of Sep. 12, 2022, and a size of 2003 kilobytes. The ST26Sequence Listing is part of the specification and incorporated in itsentirety by reference herein.

FIELD OF THE INVENTION

The present disclosure provides engineered transaminase polypeptidesuseful under industrial process conditions for the production ofpharmaceutical and fine chemical amine compounds.

BACKGROUND

Transaminases (E.C. 2.6.1) catalyze the transfer of an amino group, apair of electrons, and a proton from an amino donor compound to the ketogroup of an amino acceptor compound. Transaminase reactions can resultin the formation of a chiral amine product compound. As shown in Scheme1, an amino acceptor compound (B) (which is the keto substrate precursorof a desired chiral amine product (D)) is reacted with an amino donorcompound (A) in the presence of a transaminase. The transaminasecatalyzes the transfer of the primary amine group of the amino donorcompound (A) to the keto group of the amino acceptor compound (B). Thetransaminase reaction results in a chiral amine product compound (D)(assuming R³ is not the same as R⁴) and a new amino acceptor byproduct(or “carbonyl byproduct”) compound (C) which has a keto group.

Chiral amine compounds are frequently used in the pharmaceutical,agrochemical and chemical industries as intermediates or synthons forthe preparation of wide range of commercially desired compounds, such ascephalosporine or pyrrolidine derivatives. Typically these industrialapplications of chiral amine compounds involve using only one particularstereomeric form of the molecule (e.g., only the (R) or the (S)enantiomer is physiologically active). Transaminases are highlystereoselective and have many potential industrial uses for thesynthesis of optically pure chiral amine compounds.

Examples of the uses of transaminases to make chiral amine compoundsinclude: the enantiomeric enrichment of amino acids (See e.g., Shin etal., Biosci. Biotechnol. Biochem., 65:1782-1788 [2001]; Iwasaki et al.,Biotech. Lett., 25:1843-1846 [2003]; Iwasaki et al., Appl. Microbiol.Biotech., 69:499-505 [2004]; Yun et al., Appl. Environ. Microbiol.,70:2529-2534 [2004]; and Hwang et al., Enz. Microbiol. Technol.,34:429-426 [2004]); the preparation of intermediates and precursors ofpregabalin (e.g., WO 2008/127646); the enzymatic transamination ofcyclopamine analogs (e.g., WO 2011/017551); the stereospecific synthesisand enantiomeric enrichment of β-amino acids (e.g., WO 2005/005633); theenantiomeric enrichment of amines (See, e.g., U.S. Pat. Nos. 4,950,606;5,300,437; and 5,169,780); the production of amino acids and derivatives(See e.g., U.S. Pat. Nos. 5,316,943; 4,518,692; 4,826,766; 6,197,558;and 4,600,692); and in the production of the pharmaceutical compounds,sitagliptin, rivastigmine, and vernakalant (See e.g., U.S. Pat. No.8,293,507; Savile, et al., Sci., 329: 305-9 [2010]; WO2011/159910; andWO2012/024104).

Wild-type transaminases having the ability to catalyze a reaction ofScheme 1 have been isolated from various microorganisms, including, butnot limited to, Alcaligenes denitrificans, Bordetella bronchiseptica,Bordetella parapertussis, Brucella melitensis, Burkholderia malle,Burkholderia pseudomallei, Chromobacterium violaceum, Oceanicolagranulosus HTCC2516, Oceanobacter sp. RED65, Oceanospirillum sp. MED92,Pseudomonas putida, Ralstonia solanacearum, Rhizobium meliloti,Rhizobium sp. (strain NGR234), Bacillus thuringensis, Klebsiellapneumonia, Vibrio fluvialis (See e.g., Shin et al., Biosci. Biotechnol,Biochem., 65:1782-1788 [2001]), and Arthrobacter sp. KNK168 (See e.g.,Iwasaki et al., Appl. Microbiol. Biotechnol., 69: 499-505 [2006]; andU.S. Pat. No. 7,169,592). Several of these wild-type transaminase genesand encoded polypeptides have been sequenced (e.g., Ralstoniasolanacearum [Genbank Acc. No. YP_002257813.1, GI:207739420],Burkholderia pseudomallei 1710b [Genbank Acc. No. ABA47738.1,GI:76578263], Bordetella petrii [Genbank Acc. No. AM902716.1, GI:163258032], Vibrio fluvialis JS17 [Genbank Acc. No. AEA39183.1, GI:327207066], and Arthrobacter sp. KNK168 [GenBank Acc. No. BAK39753.1,GI:336088341]). At least two wild-type transaminases of classes EC2.6.1.18 and EC 2.6.1-19, have been crystallized and structurallycharacterized (See e.g., Yonaha et al., Agric. Biol. Chem., 47:2257-2265[1983]).

Transaminases are known that have (R)-selective or (S)-selectivestereoselectivity. For example, the wild-type transaminase fromArthrobacter sp. KNK168 is considered (R)-selective and producesprimarily (R)-amine compounds from certain substrates (See e.g., Iwasakiet al., Appl. Microbiol. Biotechnol., 69: 499-505 [2006]; and U.S. Pat.No. 7,169,592), whereas the wild-type transaminase from Vibrio fluvialisJS17 is considered (S)-selective and produces primarily (S)-aminecompounds from certain substrates (See e.g., Shin et al., Appl.Microbiol. Biotechnol., 61: 463-471 [2003]).

Non-naturally occurring transaminases having (R)-selectivity, increasedsolvent and thermal stability, and other improved properties for theconversion of a wide range of amino acceptor substrates, have beengenerated by mutagenesis and/or directed evolution of wild-type andother engineered transaminase backbone sequences (See e.g., U.S. Pat.No. 8,293,507 B2; WO2011/005477A1; WO2012/024104; and Savile et al.,Sci., 329:305-9 [2010]).

However, transaminases generally have properties that are undesirablefor commercial application in the preparation of chiral amine compounds,such as instability to industrially useful process conditions (e.g.,solvent, temperature), poor recognition of, and stereoselectivity for,commercially useful amino acceptor and/or amino donor substrates, andlow product yields due to unfavorable reaction equilibrium. Thus, thereis a need for engineered transaminases that can be used in industrialprocesses for preparing chiral amines compounds in an optically activeform.

SUMMARY OF THE INVENTION

The present disclosure provides engineered polypeptides havingtransaminase activity, polynucleotides encoding the polypeptides,methods of making the polypeptides, and methods of using thepolypeptides for the biocatalytic conversion of amino acceptor substratecompounds (i.e., keto group containing compounds) to chiral amineproduct compounds. The transaminase polypeptides of the presentdisclosure have been engineered to have one or more residue differencesas compared to a previously engineered transaminase polypeptide (ofamino acid sequence SEQ ID NO: 4) and associated enhanced solvent andthermal stability relative to the transaminase of SEQ ID NO: 4 and thewild-type transaminase of SEQ ID NO: 2. The amino acid residuedifferences are located at residue positions that result in improvementof various enzyme properties, including among others, activity,stereoselectivity, stability, expression, and product tolerance.

In particular, the engineered transaminase polypeptides of the presentdisclosure have been engineered for efficient conversion of thesubstrate,(R)-2¹-(difluoromethyl)-5-methyl-2¹H-3-aza-1(4,2)-pyridina-2(2,3)-pyrrolacyclononaphane-4,9-dione(referred to herein as “compound (1)”) to its corresponding chiral amineproduct compound,(5R,9S)-9-amino-2¹-(difluoromethyl)-5-methyl-2¹H-3-aza-1(4,2)-pyridina-2(2,3)-pyrrolacyclononaphan-4-one(referred to herein as “compound (2)”) as shown in Scheme 2.

In some embodiments, the present disclosure provides engineeredtransaminases comprising polypeptide sequences having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NOS: 4, 8, 366, and/or 650, or a functionalfragment thereof, wherein said engineered transaminases comprise atleast one substitution or substitution set in said polypeptidesequences, and wherein the amino acid positions of said polypeptidesequences are numbered with reference to SEQ ID NO: 2, 4, 8, 366, and/or650. In some embodiments, the engineered transaminase comprises apolypeptide sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 4, wherein said engineered transaminase comprises at leastone substitution or substitution set in said polypeptide sequence at oneor more positions selected from 18, 20, 21, 21/23/56/146,21/23/56/146/432, 21/23/146/417, 21/23/395/417/432, 21/53/56, 21/53/417,21/56/395, 21/417/432, 23, 23/53, 23/53/56, 23/53/56/146/395, 23/53/395,23/53/417, 23/53/432, 23/56, 23/56/395, 23/56/395/417, 23/395/417,23/417, 23/417/432, 53, 53/56, 53/146/417, 53/395, 56,56/74/241/286/314/316/323, 56/86/163/314/316/383/414/416/422,56/86/286/314/414/416, 56/86/314/316/323/394/414/422, 56/146/417,56/146/432, 56/147, 56/163, 56/163/286/316/323/383/394,56/286/314/316/323/422, 56/286/383, 56/323, 56/323/383, 56/323/383/394,56/383, 56/395, 74/81/286/316/323/383, 74/85/86/163/286/316/323/394,74/85/314/316/414/416, 74/86/163/316, 74/86/316/323/383/394,74/88/286/316/323/383, 74/88/323/383, 74/163/286/316/383/394/416,74/163/314/316, 74/163/314/316/323/394, 74/163/314/323/383/414/416,74/286, 74/286/316/323, 74/286/394/416, 74/314/323/383/394/414,74/316/323/394, 85/86/88/163/323/383/394, 85/86/163/314/323/394/414,85/286, 85/286/323, 86, 86/88/163/323/383/414/422, 86/383/394, 88,88/163/286/383, 88/286/316/323, 88/286/316/323/383/414/416, 88/316/323,146, 146/147/395/417, 146/395, 146/395/417, 146/417, 147/395/417/432,147/417, 149, 157, 163, 163/222/286/316/323/383/394, 163/286,163/286/314/316/323/414/416, 163/286/314/323/394,163/286/316/323/394/416, 163/286/414, 163/314/316/394, 163/314/323/394,163/314/383, 163/314/414, 163/316/323, 163/323, 163/383, 164, 199/417,259, 260, 284, 286, 286/314/323/383, 286/314/394,286/316/323/383/414/416, 286/316/383/394, 286/316/394/414/416, 286/323,286/323/383/414, 286/323/416, 286/383, 286/416, 314/316, 314/316/323,314/316/323/383/422, 314/316/323/394, 314/316/394, 314/323/383/394,314/383, 314/383/414/422, 315, 316, 316/323/383/394,316/323/394/414/416, 316/414/422, 323, 323/383, 323/383/394/414/416,323/394, 383, 395, 395/417, 395/417/432, 400, 401, 403, 404, 405, 406,408, 415, 417, 417/432, 420, and 422, and wherein the amino acidpositions of said polypeptide sequence are numbered with reference toSEQ ID NO: 4. In some additional embodiments, the engineeredtransaminase comprises a polypeptide sequence comprising at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to SEQ ID NO: 4, wherein said engineeredtransaminase comprises at least one substitution or substitution set insaid polypeptide sequence at one or more positions selected from74/81/286/316/323/383, 163/286/314/316/323/414/416, 163/286/314/323/394,286/314/323/383, 286/316/323/383/414/416, 315, and 408, and wherein theamino acid positions of said polypeptide sequence are numbered withreference to SEQ ID NO: 4. In some further embodiments, the engineeredtransaminase comprises a polypeptide sequence comprising at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to SEQ ID NO: 8, wherein said engineeredtransaminase comprises at least one substitution or substitution set insaid polypeptide sequence at one or more positions selected from 5,18/23/149/260/383/395/401/416, 18/23/149/383, 18/163/164, 21,21/163/315/316, 21/163/323/408, 21/408,23/56/86/149/163/164/383/401/416, 23/86, 23/149/260, 23/149/284/383/395,23/163/164/383, 23/163/164/401/416, 24, 42, 42/110, 42/187/272,42/187/324/363/366, 42/187/353, 42/272/291, 42/272/291/363,42/272/324/363/366, 42/272/363/410, 42/272/410, 42/291/313/363/410,42/291/363, 42/291/363/366, 42/353, 42/363, 46, 66, 77,86/149/163/164/383/395/401, 86/149/395, 86/163/164/260/383, 86/383, 107,110, 110/187, 110/187/253/410, 134, 138, 149/164/260/383/395/401,149/260/383, 149/416, 163/259/323/408, 163/259/408, 163/315/316,164/260/401, 164/316/383/401, 167, 186, 187, 187/253/363/366,187/272/324/363/410, 187/272/363, 187/272/363/366/410, 187/291, 189,191, 195, 199, 203, 210, 211, 248, 259/307, 260/395/401, 272, 272/353,272/363/366, 272/410, 277, 291, 305, 309, 315, 342, 343, 351, 354, 358,361, 362, 363, 363/366, 365, 367, 383, 383/401, 383/416/422, 385, 388,389, 392, 395, 396, 401, 404, 405, 408, 410, 416, 417, 439, 443, 447,450, and 451, and wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NO: 8. In yet someadditional embodiments, the engineered transaminase comprises apolypeptide sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 8, wherein said engineered transaminase comprises at leastone substitution or substitution set in said polypeptide sequence at oneor more positions selected from 18/23/149/383, 21/163/323/408, 272, 291,and 383, and wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NO: 8. In some additionalembodiments, the engineered transaminase comprises a polypeptidesequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO: 366, wherein said engineered transaminase comprises at least onesubstitution or substitution set in said polypeptide sequence at one ormore positions selected from 24, 24/42/66/291, 24/42/291/362,24/66/163/191/362/383/388, 24/66/191/199/260/291/351, 24/66/191/199/291,24/66/191/260/408, 24/66/260/291/383/388/408, 24/66/291/342/383,24/66/291/365, 24/66/342/365/388/408, 24/77/291,24/107/163/191/291/351/383/388, 24/107/291/351/365/388, 24/163/351/383,24/191/291/365, 24/199/260/351/362/383, 24/199/260/362/383/388,24/260/362/383/388, 24/291, 24/291/342/351/383, 24/291/362/388,24/291/408, 24/383/388, 24/388, 25, 28, 33, 42/191/408, 42/199/291/383,42/291/351/362/365/383/388, 42/291/351/362/383/408, 42/291/383/388,66/82/291/383, 66/163/191/365/383, 66/199/351/383, 66/291,66/291/362/365/383, 66/291/383/388, 66/383, 77/291, 77/383/388, 86,107/191/199/365/383/388, 107/191/291/383, 148, 153,163/291/362/365/383/388, 163/291/383/388, 163/383, 191/199/365/383/388,191/260/388, 191/291, 191/291/342/362/365, 191/351/383/388, 199/260/383,199/291, 260, 260/291/365/383/408, 260/365/383, 291, 291/351/383/388,291/351/383/388/408, 291/362/365, 291/365/388, 291/383, 314, 315, 316,319, 342/362, 351/383/388, 362, 362/388, 383, 383/388, 396, 397, 405,406, 413, 419, and 423, and wherein the amino acid positions of saidpolypeptide sequence are numbered with reference to SEQ ID NO: 366. Insome further embodiments, the engineered transaminase comprises apolypeptide sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO: 366, wherein said engineered transaminase comprises atleast one substitution or substitution set in said polypeptide sequenceat one or more positions selected from 24/66/191/199/291, 24/66/291/365,163/291/362/365/383/388, 163/291/383/388, 191/291/342/362/365, 291, and291/383, and wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NO: 366. In yet somefurther embodiments, the engineered transaminase comprises a polypeptidesequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO: 650, wherein said engineered transaminase comprises at least onesubstitution or substitution set in said polypeptide sequence at one ormore positions selected from 10, 13, 13/24/108/163, 13/24/108/163/311,13/24/133/199/311, 13/24/163, 13/24/199/311, 13/108, 13/108/199,13/108/311, 13/199, 13/311, 14, 14/24/108, 14/24/108/133, 14/24/108/199,14/24/199, 14/108, 14/108/133/311, 14/108/311, 14/311, 24, 24/163,24/163/199, 35, 72, 73, 78, 95, 101, 108, 108/199, 114, 154, 163, 169,175/316, 199, 199/311, 226, 293, 311, 316, 382, 383, and 386, andwherein the amino acid positions of said polypeptide sequence arenumbered with reference to SEQ ID NO: 650. In still some additionalembodiments, the engineered transaminase comprises a polypeptidesequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNO: 650, wherein said engineered transaminase comprises at least onesubstitution or substitution set in said polypeptide sequence at one ormore positions selected from 14/108/133/311, 24/163/199, 72, 78, 316,and 383, and wherein the amino acid positions of said polypeptidesequence are numbered with reference to SEQ ID NO: 650.

In some further embodiments, the engineered transaminase comprises apolypeptide sequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical to thesequence of at least one engineered transaminase variant set forth inTable 2-1, 2-2, 3-1, 3-2, 4-1, 4-2, 5-1, and/or 5-2. In yet someadditional embodiments, the engineered transaminase is a variantengineered transaminase provided in Table 2-1, 2-2, 3-1, 3-2, 4-1, 4-2,5-1, and/or 5-2. In some further embodiments, the engineeredtransaminase comprises a polypeptide sequence that is at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to the sequence of at least one engineered transaminasevariant set forth in SEQ ID NOS: 2, 4, 8, 366, and/or 650. In someadditional embodiments, the engineered transaminase comprises apolypeptide sequence comprising SEQ ID NOS: 2, 4, 8, 366, and/or 650. Insome further embodiments, the engineered transaminase comprises apolypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequenceof at least one engineered transaminase variant set forth in the evennumbered sequences of SEQ ID NOS: 6-936. In yet some additionalembodiments, the engineered transaminase comprises a polypeptidesequence set forth in the even numbered sequences of SEQ ID NOS: 6-936.In some further embodiments, the engineered transaminase comprises atleast one improved property compared to wild-type V. fluvialistransaminase. In some additional embodiments, the improved property ofthe engineered transaminase comprises improved activity on a substrate.In some further embodiments, the substrate comprises compound (1). Inyet some additional embodiments, the improved property of the engineeredtransaminase comprises improved thermostability. In some additionalembodiments, the engineered transaminase is purified. The presentdisclosure also provides compositions comprising an engineeredtransaminase provided herein. In some embodiments, the compositionscomprise more than one engineered transaminase provided herein.

The present disclosure also provides polynucleotide sequences encodingat least one engineered transaminase provided herein. In someembodiments, the polynucleotide sequence encodes at least one engineeredtransaminase, said polynucleotide sequence comprising at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NOS: 3, 7, 365, and/or 649, wherein thepolynucleotide sequence of said engineered transaminase comprises atleast one substitution at one or more positions. In some furtherembodiments, the polynucleotide sequence encodes at least one engineeredtransaminase comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNOS: 2, 4, 8, 366, and/or 650, or a functional fragment thereof. In yetsome additional embodiments, the polynucleotide sequence is operablylinked to a control sequence. In still some further embodiments, thepolynucleotide sequence is codon optimized.

The present disclosure also provides expression vectors comprising atleast one polynucleotide sequence encoding an engineered transaminaseprovided herein. In some embodiments, the expression vector comprises atleast one polynucleotide sequence comprising at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NOS: 3, 7, 365, and/or 649, wherein thepolynucleotide sequence of said engineered transaminase comprises atleast one substitution at one or more positions. In some embodiments,the expression vector comprises a polynucleotide sequence encoding atleast one engineered transaminase comprising at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NOS: 3, 7, 365, and/or 649, or a functionalfragment thereof.

The present disclosure also provides host cells comprising at least oneexpression vector provided herein. In some embodiments, the host cellcomprises at least one polynucleotide sequence provided herein. In someembodiments, the host cell comprises at least one polynucleotidesequence comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ IDNOS: 3, 7, 365, and/or 649, wherein the polynucleotide sequence encodingthe engineered transaminase comprises at least one substitution at oneor more positions. In some embodiments, the host cell comprises apolynucleotide sequence encoding at least one engineered transaminasecomprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NOS: 2, 4,8, 366, and/or 650, or a functional fragment thereof. In someembodiments, at least one polynucleotide sequence encoding an engineeredtransaminase is present in at least one expression vector.

The present disclosure also provides methods of producing an engineeredtransaminase in a host cell, comprising culturing the host cell providedherein under suitable conditions, such that at least one engineeredtransaminase is produced. In some embodiments, the methods furthercomprise recovering at least one engineered transaminase from theculture and/or host cell. In some additional embodiments, the methodsfurther comprise the step of purifying said at least one engineeredtransaminase.

In some embodiments, the engineered polypeptide having transaminaseactivity is immobilized on a solid support, optionally wherein the solidsupport is selected from a bead or resin comprising polymethacrylatewith epoxide functional groups, polymethacrylate with amino epoxidefunctional groups, styrene/DVB copolymer or polymethacrylate withoctadecyl functional groups.

In some embodiments, the engineered polypeptide having transaminaseactivity is capable of converting a substrate of compound (1) to aproduct of compound (2) under suitable reaction conditions. In someembodiments, the engineered polypeptide is capable of convertingcompound (1) to compound (2) with at least 1.2 fold, 2 fold, 5 fold, 10fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, or greater than theactivity of are reference sequence SEQ ID NO: 2, 4, 8, 366, and/or 650),under suitable reaction conditions. In some embodiments, the engineeredpolypeptide is capable of converting compound (1) to compound (2) withincreased activity relative to a reference sequence (e.g., SEQ ID NO: 2,4, 8, 366, and/or 650), in which the suitable reaction conditionscomprise compound (1) at a loading of at least 100 g/L, about 1 g/Lengineered polypeptide, about 0.5 g/L PLP, about 1 M isopropylamine,about pH 9, and about 50° C.

In some embodiments, the present disclosure provides a process ofpreparing compound (2)

comprising a step of contacting a substrate of compound (1)

with an engineered polypeptide as disclosed herein in the presence of anamino group donor under suitable reaction conditions.

In some embodiments, the processes using the engineered polypeptidesdisclosed herein can be carried out wherein the compound (2) is producedin at least 90%, 97%, 98%, 99% or greater enantiomeric anddiastereomeric excess.

Any of the processes disclosed herein using the engineered polypeptidesfor the preparation of compound (2) can be carried out under a range ofsuitable reaction conditions, including but not limited to, ranges ofamine donor, pH, temperature, buffer, solvent system, substrate loading,polypeptide loading, cofactor loading, pressure, and reaction time. Forexample, in some embodiments, the preparation of compound (2) can becarried out wherein the suitable reaction conditions comprise: (a)substrate loading of about 10 to 300 g/L of substrate compound (e.g., 50g/L or 200 g/L of compound (1)); (b) of about 0.5 g/L to 60 g/Lengineered polypeptide; (c) IPM concentration of about 0.5 to 2 M; (d)PLP cofactor concentration of about 0.1 to 1 g/L; (e) DMSO concentrationof about 0% (v/v) to about 20% (v/v); (f) pH of about 8.5 to 11.5; and(g) temperature of about 45° C. to 65° C. In some embodiments, thesuitable reaction conditions comprise: (a) about 100 g/L of substratecompound (e.g., compound (1)); (b) about 1 g/L engineered polypeptide;(c) about 1 M isopropylamine (IPM); (d) about 0.5 g/L pyridoxalphosphate (PLP); (e) about pH 9; and (g) about 50° C.

In some embodiments, the processes using the engineered polypeptidesdisclosed herein can be carried out wherein the amino group donor isselected from isopropylamine, alanine, 3-aminobutyric acid, ormethylbenzylamine. In some embodiments, the amino group donor isisopropylamine.

DESCRIPTION OF THE INVENTION

For the descriptions provided herein, the use of the singular includesthe plural (and vice versa) unless specifically stated otherwise. Forinstance, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly indicates otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Both the foregoing general description, including the drawings, and thefollowing detailed description are exemplary and explanatory only andare not restrictive of this invention. Moreover, the section headingsused herein are for organizational purposes only and not to be construedas limiting the subject matter described.

Definitions

As used herein, the following terms are intended to have the followingmeanings. In reference to the present invention, the technical andscientific terms used in the descriptions herein will have the meaningscommonly understood by one of ordinary skill in the art, unlessspecifically defined otherwise. Accordingly, the following terms areintended to have the following meanings. In addition, all patents andpublications, including all sequences disclosed within such patents andpublications, referred to herein are expressly incorporated byreference.

Unless otherwise indicated, the practice of the present disclosureinvolves conventional techniques commonly used in molecular biology,fermentation, microbiology, and related fields, which are known to thoseof skill in the art. Unless defined otherwise herein, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, the preferred methods and materials are described.Indeed, it is intended that the present invention not be limited to theparticular methodology, protocols, and reagents described herein, asthese may vary, depending upon the context in which they are used. Theheadings provided herein are not limitations of the various aspects orembodiments of the present invention that can be had by reference to thespecification as a whole. Accordingly, the terms defined below are morefully defined by reference to the specification as a whole.

Nonetheless, in order to facilitate understanding of the presentdisclosure, a number of terms are defined below. Numeric ranges areinclusive of the numbers defining the range. Thus, every numerical rangedisclosed herein is intended to encompass every narrower numerical rangethat falls within such broader numerical range, as if such narrowernumerical ranges were all expressly written herein. It is also intendedthat every maximum (or minimum) numerical limitation disclosed hereinincludes every lower (or higher) numerical limitation, as if such lower(or higher) numerical limitations were expressly written herein.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

As used herein and in the appended claims, the singular “a”, “an” and“the” include the plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to a “host cell” includes aplurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation and amino acid sequences are written left to rightin amino to carboxy orientation, respectively.

As used herein, the terms “protein,” “polypeptide,” and “peptide” areused interchangeably herein to denote a polymer of at least two aminoacids covalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation, phosphorylation,lipidation, myristilation, ubiquitination, etc.). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids.

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine HIS H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

As used herein, “hydrophilic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity of lessthan zero according to the normalized consensus hydrophobicity scale ofEisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142 [1984]).Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser(S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K)and L-Arg (R).

As used herein, “acidic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pK value of lessthan about 6 when the amino acid is included in a peptide orpolypeptide. Acidic amino acids typically have negatively charged sidechains at physiological pH due to loss of a hydrogen ion. Geneticallyencoded acidic amino acids include L-Glu (E) and L-Asp (D).

As used herein, “basic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pK value ofgreater than about 6 when the amino acid is included in a peptide orpolypeptide. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

As used herein, “polar amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain that is uncharged atphysiological pH, but which has at least one bond in which the pair ofelectrons shared in common by two atoms is held more closely by one ofthe atoms. Genetically encoded polar amino acids include L-Asn (N),L-Gln (Q), L-Ser (S) and L-Thr (T).

As used herein, “hydrophobic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity ofgreater than zero according to the normalized consensus hydrophobicityscale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142[1984]). Genetically encoded hydrophobic amino acids include L-Pro (P),L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala(A) and L-Tyr (Y).

As used herein, “aromatic amino acid or residue” refers to a hydrophilicor hydrophobic amino acid or residue having a side chain that includesat least one aromatic or heteroaromatic ring. Genetically encodedaromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W).Although owing to the pKa of its heteroaromatic nitrogen atom L-His (H)it is sometimes classified as a basic residue, or as an aromatic residueas its side chain includes a heteroaromatic ring, herein histidine isclassified as a hydrophilic residue or as a “constrained residue” (seebelow).

As used herein, “constrained amino acid or residue” refers to an aminoacid or residue that has a constrained geometry. Herein, constrainedresidues include L-Pro (P) and L-His (H). Histidine has a constrainedgeometry because it has a relatively small imidazole ring. Proline has aconstrained geometry because it also has a five membered ring.

As used herein, “non-polar amino acid or residue” refers to ahydrophobic amino acid or residue having a side chain that is unchargedat physiological pH and which has bonds in which the pair of electronsshared in common by two atoms is generally held equally by each of thetwo atoms (i.e., the side chain is not polar). Genetically encodednon-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile(I), L-Met (M) and L-Ala (A).

As used herein, “aliphatic amino acid or residue” refers to ahydrophobic amino acid or residue having an aliphatic hydrocarbon sidechain. Genetically encoded aliphatic amino acids include L-Ala (A),L-Val (V), L-Leu (L) and L-Ile (I). It is noted that cysteine (or“L-Cys” or “[C]”) is unusual in that it can form disulfide bridges withother L-Cys (C) amino acids or other sulfanyl- or sulfhydryl-containingamino acids. The “cysteine-like residues” include cysteine and otheramino acids that contain sulfhydryl moieties that are available forformation of disulfide bridges. The ability of L-Cys (C) (and otheramino acids with —SH containing side chains) to exist in a peptide ineither the reduced free —SH or oxidized disulfide-bridged form affectswhether L-Cys (C) contributes net hydrophobic or hydrophilic characterto a peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29according to the normalized consensus scale of Eisenberg (Eisenberg etal., 1984, supra), it is to be understood that for purposes of thepresent disclosure, L-Cys (C) is categorized into its own unique group.

As used herein, “small amino acid or residue” refers to an amino acid orresidue having a side chain that is composed of a total three or fewercarbon and/or heteroatoms (excluding the α-carbon and hydrogens). Thesmall amino acids or residues may be further categorized as aliphatic,non-polar, polar or acidic small amino acids or residues, in accordancewith the above definitions. Genetically-encoded small amino acidsinclude L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T)and L-Asp (D).

As used herein, “hydroxyl-containing amino acid or residue” refers to anamino acid containing a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

As used herein, “amino acid difference” and “residue difference” referto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. The positions of amino acid differencesgenerally are referred to herein as “Xn,” where n refers to thecorresponding position in the reference sequence upon which the residuedifference is based. For example, a “residue difference at position X40as compared to SEQ ID NO:2” refers to a difference of the amino acidresidue at the polypeptide position corresponding to position 40 of SEQID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has ahistidine at position 40, then a “residue difference at position X40 ascompared to SEQ ID NO:2” refers to an amino acid substitution of anyresidue other than histidine at the position of the polypeptidecorresponding to position 40 of SEQ ID NO:2. In most instances herein,the specific amino acid residue difference at a position is indicated as“XnY” where “Xn” specified the corresponding position as describedabove, and “Y” is the single letter identifier of the amino acid foundin the engineered polypeptide (i.e., the different residue than in thereference polypeptide). In some instances, the present disclosure alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present disclosure can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where residuedifferences are present relative to the reference sequence. In someembodiments, where more than one amino acid can be used in a specificresidue position of a polypeptide, the various amino acid residues thatcan be used are separated by a “/” (e.g., X192A/G). In some embodiments,in which there are variants with multiple substitutions, thesubstitutions are separated by either a semicolon (;) or a slash (/)(e.g., Y17V;I259T;E347K or Y17V/I259T/E347K).

The present disclosure includes engineered polypeptide sequencescomprising one or more amino acid differences that include either/orboth conservative and non-conservative amino acid substitutions. Theamino acid sequences of the specific recombinant carbonic anhydrasepolypeptides included in the Sequence Listing of the present disclosureinclude an initiating methionine (M) residue (i.e., M represents residueposition 1). The skilled artisan, however, understands that thisinitiating methionine residue can be removed by biological processingmachinery, such as in a host cell or in vitro translation system, togenerate a mature protein lacking the initiating methionine residue, butotherwise retaining the enzyme's properties. Consequently, the term“amino acid residue difference relative to SEQ ID NO:2 at position Xn”as used herein may refer to position “Xn” or to the correspondingposition (e.g., position (X−1)n) in a reference sequence that has beenprocessed so as to lack the starting methionine.

As used herein, the phrase “conservative amino acid substitutions”refers to the interchangeability of residues having similar side chains,and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, in some embodiments,an amino acid with an aliphatic side chain is substituted with anotheraliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine);an amino acid with a hydroxyl side chain is substituted with anotheramino acid with a hydroxyl side chain (e.g., serine and threonine); anamino acids having aromatic side chains is substituted with anotheramino acid having an aromatic side chain (e.g., phenylalanine, tyrosine,tryptophan, and histidine); an amino acid with a basic side chain issubstituted with another amino acid with a basis side chain (e.g.,lysine and arginine); an amino acid with an acidic side chain issubstituted with another amino acid with an acidic side chain (e.g.,aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively. Exemplary conservative substitutions are provided inTable 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions ResiduePotential Conservative Substitutions A, L, V, I Other aliphatic (A, L,V, I) Other non-polar (A, L, V, I, G, M) G, M Other non-polar (A, L, V,I, G, M) D, E Other acidic (D, E) K, R Other basic (K, R) N, Q, S, TOther polar H, Y, W, F Other aromatic (H, Y, W, F) C, P Non-polar

As used herein, the phrase “non-conservative substitution” refers tosubstitution of an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification of the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thepolypeptide while retaining enzymatic activity and/or retaining theimproved properties of an engineered enzyme. Deletions can be directedto the internal portions and/or terminal portions of the polypeptide. Invarious embodiments, the deletion can comprise a continuous segment orcan be discontinuous.

As used herein, “insertion” refers to modification of the polypeptide byaddition of one or more amino acids to the reference polypeptide. Insome embodiments, the improved engineered transaminase enzymes compriseinsertions of one or more amino acids to the naturally occurringtransaminase polypeptide as well as insertions of one or more aminoacids to engineered transaminase polypeptides. Insertions can be in theinternal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions in a polypeptide sequence, as comparedto a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. Insome embodiments, a substitution set refers to the set of amino acidsubstitutions that is present in any of the variant transaminases listedin the Tables provided in the Examples. The term “substitution set” isalso used in reference to a group of nucleotide substitutions in apolynucleotide sequence, as compared to a reference sequence.

As used herein, “fragment” refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can typically have about 80%, about 90%, about 95%,about 98%, or about 99% of the full-length transaminase polypeptide, forexample the polypeptide of SEQ ID NO:2. In some embodiments, thefragment is “biologically active” (i.e., it exhibits the same enzymaticactivity as the full-length sequence).

As used herein, “isolated polypeptide” refers to a polypeptide that issubstantially separated from other contaminants that naturally accompanyit (e.g., proteins, lipids, and polynucleotides). The term embracespolypeptides which have been removed or purified from theirnaturally-occurring environment or expression system (e.g., host cell orin vitro synthesis). The improved transaminase enzymes may be presentwithin a cell, present in the cellular medium, or prepared in variousforms, such as lysates or isolated preparations. As such, in someembodiments, the engineered transaminase polypeptides of the presentdisclosure can be an isolated polypeptide.

As used herein, “substantially pure polypeptide” refers to a compositionin which the polypeptide species is the predominant species present(i.e., on a molar or weight basis it is more abundant than any otherindividual macromolecular species in the composition), and is generallya substantially purified composition when the object species comprisesat least about 50 percent of the macromolecular species present by moleor % weight. Generally, a substantially pure engineered transaminasepolypeptide composition comprises about 60% or more, about 70% or more,about 80% or more, about 90% or more, about 91% or more, about 92% ormore, about 93% or more, about 94% or more, about 95% or more, about 96%or more, about 97% or more, about 98% or more, or about 99% of allmacromolecular species by mole or % weight present in the composition.Solvent species, small molecules (<500 Daltons), and elemental ionspecies are not considered macromolecular species. In some embodiments,the isolated improved transaminase polypeptide is a substantially purepolypeptide composition.

As used herein, “substantially pure polynucleotide” refers to acomposition in which the polynucleotide species is the predominantspecies present (i.e., on a molar or weight basis it is more abundantthan any other individual macromolecular species in the composition),and is generally a substantially purified composition when the objectspecies comprises at least about 50 percent of the macromolecularspecies present by mole or % weight. Generally, a substantially pureengineered transaminase polynucleotide composition comprises about 60%or more, about 70% or more, about 80% or more, about 90% or more, about91% or more, about 92% or more, about 93% or more, about 94% or more,about 95% or more, about 96% or more, about 97% or more, about 98% ormore, or about 99% of all macromolecular species by mole or % weightpresent in the composition. In some embodiments, the isolated improvedtransaminase polypeptide is a substantially pure polynucleotidecomposition.

As used herein, “polynucleotide” and “nucleic acid” refer to two or morenucleosides that are covalently linked together. The polynucleotide maybe wholly comprised ribonucleosides (i.e., an RNA), wholly comprised of2′ deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), itmay include one or more modified and/or synthetic nucleobases (e.g.,inosine, xanthine, hypoxanthine, etc.). Preferably, such modified orsynthetic nucleobases will be encoding nucleobases.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

As used herein, “hybridization stringency” relates to hybridizationconditions, such as washing conditions, in the hybridization of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA; with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T, as determined under the solution condition for a definedpolynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are known to those of skill in the art.

As used herein, “coding sequence” refers to that portion of a nucleicacid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. In some embodiments, thepolynucleotides encoding the transaminase enzymes may be codon optimizedfor optimal production from the host organism selected for expression.Although the genetic code is degenerate in that most amino acids arerepresented by several codons, called “synonyms” or “synonymous” codons,it is well known that codon usage by particular organisms is nonrandomand biased towards particular codon triplets. This codon usage bias maybe higher in reference to a given gene, genes of common function orancestral origin, highly expressed proteins versus low copy numberproteins, and the aggregate protein coding regions of an organism'sgenome. In some embodiments, the polynucleotides encoding thetransaminase enzymes may be codon optimized for optimal production fromthe host organism selected for expression.

As used herein, “preferred, optimal, high codon usage bias codons”refers interchangeably to codons that are used at higher frequency inthe protein coding regions than other codons that code for the sameamino acid. The preferred codons may be determined in relation to codonusage in a single gene, a set of genes of common function or origin,highly expressed genes, the codon frequency in the aggregate proteincoding regions of the whole organism, codon frequency in the aggregateprotein coding regions of related organisms, or combinations thereof.Codons whose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (See e.g., GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney,Bioinform., 14:372-73 [1998]; Stenico et al., Nucleic Acids Res.,222:437-46 [1994]; and Wright, Gene 87:23-29 [1990]). Codon usage tablesare available for a growing list of organisms (See e.g., Wada et al.,Nucleic Acids Res., 20:2111-2118 [1992]; Nakamura et al., Nucl. AcidsRes., 28:292 [2000]; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” Neidhardt, et al. (eds.), ASM Press,Washington D.C., [1996], p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (See e.g., Uberbacher, Meth.Enzymol., 266:259-281 [1996]; Tiwari et al., Comput. Appl. Biosci.,13:263-270 [1997]).

As used herein, “control sequence” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide and/or polypeptide of the present disclosure. Eachcontrol sequence may be native or foreign to the polynucleotide ofinterest. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator.

As used herein, “operably linked” is defined herein as a configurationin which a control sequence is appropriately placed (i.e., in afunctional relationship) at a position relative to a polynucleotide ofinterest such that the control sequence directs or regulates theexpression of the polynucleotide and/or polypeptide of interest.

As used herein, “promoter sequence” refers to a nucleic acid sequencethat is recognized by a host cell for expression of a polynucleotide ofinterest, such as a coding sequence. The control sequence may comprisean appropriate promoter sequence. The promoter sequence containstranscriptional control sequences, which mediate the expression of apolynucleotide of interest. The promoter may be any nucleic acidsequence which shows transcriptional activity in the host cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell.

As used herein, “naturally occurring” and “wild-type” refers to the formfound in nature. For example, a naturally occurring or wild-typepolypeptide or polynucleotide sequence is a sequence present in anorganism that can be isolated from a source in nature and which has notbeen intentionally modified by human manipulation.

As used herein, “non-naturally occurring,” “engineered,” and“recombinant” when used in the present disclosure with reference to(e.g., a cell, nucleic acid, or polypeptide), refers to a material, or amaterial corresponding to the natural or native form of the material,that has been modified in a manner that would not otherwise exist innature. In some embodiments the material is identical to naturallyoccurring material, but is produced or derived from synthetic materialsand/or by manipulation using recombinant techniques. Non-limitingexamples include, among others, recombinant cells expressing genes thatare not found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise expressed at a different level.

As used herein, “percentage of sequence identity,” “percent identity,”and “percent identical” refer to comparisons between polynucleotidesequences or polypeptide sequences, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whicheither the identical nucleic acid base or amino acid residue occurs inboth sequences or a nucleic acid base or amino acid residue is alignedwith a gap to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Determination of optimal alignment and percentsequence identity is performed using the BLAST and BLAST 2.0 algorithms(See e.g., Altschul et al., J. Mol. Biol. 215: 403-410 [1990]; andAltschul et al., Nucl. Acids Res., 25: 3389-3402 [1977]). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length Win the querysequence, which either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as, the neighborhood word score threshold(Altschul et al., supra). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penaltyscore for mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (See e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915 [1989]).

Numerous other algorithms are available and known in the art thatfunction similarly to BLAST in providing percent identity for twosequences. Optimal alignment of sequences for comparison can beconducted using any suitable method known in the art (e.g., by the localhomology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482[1981]; by the homology alignment algorithm of Needleman and Wunsch, J.Mol. Biol., 48:443 [1970]; by the search for similarity method ofPearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; and/or bycomputerized implementations of these algorithms [GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package]), or by visualinspection, using methods commonly known in the art. Additionally,determination of sequence alignment and percent sequence identity canemploy the BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison WI), using the default parameters provided.

As used herein, “substantial identity” refers to a polynucleotide orpolypeptide sequence that has at least 80 percent sequence identity, atleast 85 percent identity and 89 to 95 percent sequence identity, moreusually at least 99 percent sequence identity as compared to a referencesequence over a comparison window of at least 20 residue positions,frequently over a window of at least 30-50 residues, wherein thepercentage of sequence identity is calculated by comparing the referencesequence to a sequence that includes deletions or additions which total20 percent or less of the reference sequence over the window ofcomparison. In specific embodiments applied to polypeptides, the term“substantial identity” means that two polypeptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 80 percent sequence identity, preferably atleast 89 percent sequence identity, at least 95 percent sequenceidentity or more (e.g., 99 percent sequence identity). In some preferredembodiments, residue positions that are not identical differ byconservative amino acid substitutions.

As used herein, “reference sequence” refers to a defined sequence towhich another sequence is compared. A reference sequence may be a subsetof a larger sequence, for example, a segment of a full-length gene orpolypeptide sequence. Generally, a reference sequence is at least 20nucleotide or amino acid residues in length, at least 25 residues inlength, at least 50 residues in length, or the full length of thenucleic acid or polypeptide. Since two polynucleotides or polypeptidesmay each (1) comprise a sequence (i.e., a portion of the completesequence) that is similar between the two sequences, and (2) may furthercomprise a sequence that is divergent between the two sequences,sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides over a comparison window to identify and compare localregions of sequence similarity. The term “reference sequence” is notintended to be limited to wild-type sequences, and can includeengineered or altered sequences. For example, in some embodiments, a“reference sequence” can be a previously engineered or altered aminoacid sequence.

As used herein, “comparison window” refers to a conceptual segment of atleast about 20 contiguous nucleotide positions or amino acids residueswherein a sequence may be compared to a reference sequence of at least20 contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “corresponding to,” “reference to,” and “relative to”when used in the context of the numbering of a given amino acid orpolynucleotide sequence refers to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence. For example, a given amino acid sequence, such as that of anengineered transaminase, can be aligned to a reference sequence byintroducing gaps to optimize residue matches between the two sequences.In these cases, although the gaps are present, the numbering of theresidue in the given amino acid or polynucleotide sequence is made withrespect to the reference sequence to which it has been aligned. As usedherein, a reference to a residue position, such as “Xn” as furtherdescribed below, is to be construed as referring to “a residuecorresponding to”, unless specifically denoted otherwise. Thus, forexample, “X94” refers to any amino acid at position 94 in a polypeptidesequence.

As used herein, when used in reference to a nucleic acid or polypeptide,the term “heterologous” refers to a sequence that is not normallyexpressed and secreted by an organism (e.g., a wild-type organism). Insome embodiments, the term encompasses a sequence that comprises two ormore subsequences which are not found in the same relationship to eachother as normally found in nature, or is recombinantly engineered sothat its level of expression, or physical relationship to other nucleicacids or other molecules in a cell, or structure, is not normally foundin nature. For instance, a heterologous nucleic acid is typicallyrecombinantly produced, having two or more sequences from unrelatedgenes arranged in a manner not found in nature (e.g., a nucleic acidopen reading frame (ORF) of the disclosure operatively linked to apromoter sequence inserted into an expression cassette, such as avector). In some embodiments, “heterologous polynucleotide” refers toany polynucleotide that is introduced into a host cell by laboratorytechniques, and includes polynucleotides that are removed from a hostcell, subjected to laboratory manipulation, and then reintroduced into ahost cell.

As used herein, “improved enzyme property” refers to a transaminase thatexhibits an improvement in any enzyme property as compared to areference transaminase. For the engineered transaminase polypeptidesdescribed herein, the comparison is generally made to the wild-typetransaminase enzyme, although in some embodiments, the referencetransaminase can be another improved engineered transaminase. Enzymeproperties for which improvement is desirable include, but are notlimited to, enzymatic activity (which can be expressed in terms ofpercent conversion of the substrate at a specified reaction time using aspecified amount of transaminase), chemoselectivity, thermal stability,solvent stability, pH activity profile, cofactor requirements,refractoriness to inhibitors (e.g., product inhibition),stereospecificity, and stereoselectivity (including enantioselectivity).

As used herein, “increased enzymatic activity” and “increased activity”refer to an improved property of an engineered enzyme, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of transaminase) as compared to a reference enzyme asdescribed herein. Any property relating to enzyme activity may beaffected, including the classical enzyme properties of K_(m), V_(max) ork_(cat), changes of which can lead to increased enzymatic activity.Comparisons of enzyme activities are made using a defined preparation ofenzyme, a defined assay under a set condition, and one or more definedsubstrates, as further described in detail herein. Generally, whenenzymes in cell lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

As used herein, “conversion” refers to the enzymatic transformation of asubstrate to the corresponding product.

As used herein “percent conversion” refers to the percent of thesubstrate that is converted to the product within a period of time underspecified conditions. Thus, for example, the “enzymatic activity” or“activity” of a transaminase polypeptide can be expressed as “percentconversion” of the substrate to the product.

As used herein, “chemoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one product over another.

As used herein, “thermostable” and “thermal stable” are usedinterchangeably to refer to a polypeptide that is resistant toinactivation when exposed to a set of temperature conditions (e.g.,40-80° C.) for a period of time (e.g., 0.5-24 hrs) compared to theuntreated enzyme, thus retaining a certain level of residual activity(e.g., more than 60% to 80%) after exposure to elevated temperatures.

As used herein, “solvent stable” refers to the ability of a polypeptideto maintain similar activity (e.g., more than 60% to 80%) after exposureto varying concentrations (e.g., 5-99%) of solvent (e.g., isopropylalcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,butylacetate, methyl tert-butylether, etc.) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme.

As used herein, “pH stable” refers to a transaminase polypeptide thatmaintains similar activity (e.g., more than 60% to 80%) after exposureto high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme.

As used herein, “thermo- and solvent stable” refers to a transaminasepolypeptide that is both thermostable and solvent stable.

As used herein, “suitable reaction conditions” refer to those conditionsin the biocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, temperature, pH, buffers,co-solvents, etc.) under which a transaminase polypeptide of the presentdisclosure is capable of transamination. Exemplary “suitable reactionconditions” are provided in the present disclosure and illustrated bythe Examples.

As used herein, “loading,” such as in “compound loading,” “enzymeloading,” or “cofactor loading” refers to the concentration or amount ofa component in a reaction mixture at the start of the reaction.

As used herein, “substrate” in the context of a biocatalyst mediatedprocess refers to the compound or molecule acted on by the biocatalyst.

As used herein “product” in the context of a biocatalyst mediatedprocess refers to the compound or molecule resulting from the action ofthe biocatalyst.

As used herein, “equilibration” as used herein refers to the processresulting in a steady state concentration of chemical species in achemical or enzymatic reaction (e.g., interconversion of two species Aand B), including interconversion of stereoisomers, as determined by theforward rate constant and the reverse rate constant of the chemical orenzymatic reaction.

“Transaminase” or “aminotransferase” are used interchangeably herein torefer to a polypeptide having an enzymatic capability of transferring anamino group (—NH₂), a pair of electrons, and a proton from the primaryamine of an amine donor compound to the carbonyl group (C═O) of an amineacceptor compound, thereby converting the amine donor compound into itscorresponding carbonyl compound and the carbonyl acceptor compound intoits corresponding primary amine compound (See e.g., Scheme 1).Transaminases as used herein include naturally occurring (wild type)transaminase as well as non-naturally occurring engineered polypeptidesgenerated by human manipulation.

“Amino group donor” or “amino donor” used interchangeably herein torefer to an amino group containing compound which is capable of donatingan amino group to an acceptor carbonyl compound (i.e., an amino groupacceptor), thereby becoming a carbonyl by-product. Amino group donorshave the general structural formula,

in which each of R¹, and R², when taken independently, is an alkyl, analkylaryl group, or aryl group which is unsubstituted or substitutedwith one or more enzymatically non-inhibiting groups. R¹ can be the sameor different from R² in structure or chirality. The groups R¹ and R²,taken together, may form a ring that is unsubstituted, substituted, orfused to other rings. Typical amino group donors include chiral andachiral amino acids, and chiral and achiral amines.

“Chiral amine” refers to an amino group containing compound having thegeneral structural formula,

in which each of R¹, and R², when taken independently, is an alkyl, analkylaryl group, or aryl group which is unsubstituted or substitutedwith one or more groups. R¹ is different from R² in structure causingthe carbon bearing the amino group (denoted with a *) to be stereogeniccenter. The groups R¹ and R², taken together, may form a ring that isunsubstituted, substituted, or fused to other rings but is otherwise notsymmetrical.

“Carbonyl by-product” refers to the carbonyl compound formed from theamino group donor when the amino group on the amino group donor istransferred to the amino group acceptor in a transamination reaction.The carbonyl by-product has the general structural formula,

wherein R¹ and R² are defined above for the amino group donor.

“Amino acceptor” and “amine acceptor,” “keto substrate,” are usedinterchangeably herein to refer to a carbonyl group containing compoundthat accepts the amino group from an amino group donor in a reactionmediated by a transaminase (See e.g., Scheme 1). In the context of thepresent disclosure, the amino acceptor compound for the transaminase caninclude, among others, compound (2).

“Cofactor,” as used herein, refers to a non-protein compound thatoperates in combination with an enzyme in catalyzing a reaction. As usedherein, “cofactor” is intended to encompass the vitamin B₆ familycompounds PLP, PN, PL, PM, PNP, and PMP, which are sometimes alsoreferred to as coenzymes.

“Pyridoxal-phosphate,” “PLP,” “pyridoxal-5′-phosphate,” “PYP,” and “P5P”are used interchangeably herein to refer to the compound that acts as acofactor in transaminase reactions. In some embodiments, pyridoxalphosphate is defined by the structure1-(4′-formyl-3′-hydroxy-2′-methyl-5′-pyridyl)methoxyphosphonic acid, CASnumber [54-47-7]. Pyridoxal-5′-phosphate can be produced in vivo byphosphorylation and oxidation of pyridoxol (also known as Vitamin B₆).In transamination reactions using transaminase enzymes, the amine groupof the amino donor is transferred to the cofactor to produce a ketobyproduct, while pyridoxal-5′-phosphate is converted to pyridoxaminephosphate. Pyridoxal-5′-phosphate is regenerated by reaction with adifferent keto compound (the amino acceptor). The transfer of the aminegroup from pyridoxamine phosphate to the amino acceptor produces anamine and regenerates the cofactor. In some embodiments, thepyridoxal-5′-phosphate can be replaced by other members of the vitaminB₆ family, including pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM),and their phosphorylated counterparts; pyridoxine phosphate (PNP), andpyridoxamine phosphate (PMP).

“Alkyl” refers to groups of from 1 to 18 carbon atoms, either straightchained or branched, particularly from 1 to 8 carbon atoms, and moreparticularly 1 to 6 carbon atoms. An alkyl with a specified number ofcarbon atoms is denoted in parenthesis (e.g., (C1-C4) alkyl refers to analkyl of 1 to 4 carbon atoms).

“Alkenyl” refers to groups of from 2 to 12 carbon atoms, either straightor branched containing at least one double bond but optionallycontaining more than one double bond.

“Alkynyl” refers to groups of from 2 to 12 carbon atoms, either straightor branched containing at least one triple bond but optionallycontaining more than one triple bond, and optionally containing one ormore double bonded moieties.

“Aryl” refers to an unsaturated aromatic carbocyclic group of from 5 to14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl). For multiple condensedrings, at least one of the rings is aromatic. Representative arylsinclude phenyl, pyridyl, naphthyl and the like.

“Arylalkyl” refers to an alkyl substituted with an aryl moiety.Representative arylalkyl groups include benzyl, phenethyl and the like.

“Arylalkenyl” refers to an alkenyl as defined herein substituted with anaryl group.

“Arylalkynyl” refers to an alkynyl as defined herein substituted with anaryl group.

“Heteroaryl” refers to an aromatic heterocyclic group of 5 to 14 ringatoms containing 1 to 4 ring heteroatoms selected from oxygen, nitrogenand sulfur within the ring. Heteroaryl groups can have a single ring(e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinylor benzothienyl). For multiple condensed rings, at least one of therings is aromatic.

“Heteroarylalkyl” refers to an alkyl substituted with a heteroarylmoiety as defined herein.

“Heteroarylalkenyl” refers to an alkenyl substituted with a heteroarylgroup as defined herein.

“Heteroarylalkynyl” refers to an alkynyl substituted with a heteroarylmoiety as defined herein.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 12 carbon atomshaving a single cyclic ring or multiple condensed rings. Representativecycloalkyl groups include, by way of example, single ring structuressuch as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl,1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and thelike, or multiple ring structures, including bridged ring systems, suchas adamantyl, and the like.

“Heterocycle” and interchangeably “heterocycloalkyl” refer to asaturated or unsaturated group having a single ring or multiplecondensed rings, from 3 to 14 ring atoms having from 1 to 4 hetero atomsselected from nitrogen, sulfur or oxygen within the ring. Heterocyclicgroups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) ormultiple condensed rings (e.g., indolinyl, dihydrobenzofuran orquinuclidinyl). Representative heterocycles and heteroaryls include, butare not limited to, furan, thiophene, thiazole, oxazole, pyrrole,imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine,indolizine, isoindole, indole, indazole, purine, quinolizine,isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline,quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine,acridine, phenanthroline, isothiazole, phenazine, isoxazole,phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine,piperazine, pyrrolidine, indoline and the like.

“Cycloalkylalkyl” refers to an alkyl substituted with a cycloalkylmoiety as defined herein.

“Cycloalkylalkenyl” refers to an alkenyl substituted with a cycloalkylmoiety as defined herein.

“Cycloalkylalkynyl” refers to an alkynyl substituted with a cycloalkylmoiety as defined herein.

“Heterocycloalkylalkyl” refers to an alkyl substituted with aheterocycloalkyl moiety as defined herein.

“Heterocycoalkenyl” refers to an alkenyl substituted with aheterocycloalkyl moiety as defined herein.

“Heterocycloalkylalkynyl” refers to an alkynyl substituted with aheterocycloalkyl moiety as defined herein.

“Alkoxy” or “Alkyloxy” refers to the group alkyl-O— wherein the alkylgroup is as defined above, including optionally substituted alkyl groupsas also defined above.

“Amino” refers to the group —NH₂. Substituted amino refers to the group—NHR′, NR′R′, and NR′R′R′, where each R′ is independently of the othersselected from substituted or unsubstituted alkyl, cycloalkyl,heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, alkyloxy,aryl, heteroaryl, arylalkyl, heteroarylalkyl, acyl, alkyloxycarbonyl,sulfanyl, sulfinyl, sulfonyl, and the like. Typical amino groupsinclude, but are not limited to, dimethylamino, diethylamino,trimethylammonium, triethylammonium, methylysulfonylamino,furanyl-oxy-sulfamino, and the like.

“Carboxy” refers to —COOH.

“Carbonyl” refers to —C(O)—, which may have a variety of substituents toform different carbonyl groups including acids, acid halides, aldehydes,amides, esters, and ketones.

“Hydroxy” refers to —OH.

“Cyano” refers to —CN.

“Halogen” or “halo” refers to fluoro, chloro, bromo and iodo.

“Sulfonyl” refers to —SO₂—. Substituted sulfonyl refers to —SO₂R′, whereR′ is a suitable substituent as described below.

“Fused” or “fused rings” such as in fused aryl or fused heteroarylrefers to two or more rings joined such that they have at least two ringatoms in common. Fused aryl refers to fused rings in which at least oneof the rings is an aryl. Fused heteroaryl refers to fused rings in whichat least one of the rings is a heteroaryl.

“Substituted” unless otherwise specified, refers to replacement ofpositions occupied by hydrogen in the foregoing groups with substituentsexemplified by, but not limited to, hydroxy, oxo, nitro, methoxy,ethoxy, alkyloxy, substituted alkyloxy, trifluoromethoxy, haloalkyloxy,fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl,alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl,hydroxyalkyl, alkyloxyalkyl, thio, alkylthio, acyl, carboxy,alkyloxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl,alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido,cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl,acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substitutedaryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl,heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl,substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl,morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; andpreferred heteroatoms are oxygen, nitrogen, and sulfur. It is understoodthat where open valences exist on these substituents they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further understood that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present disclosure, and is otherwise chemically reasonable.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. One of ordinary skill in the art would understand that withrespect to any molecule described as containing one or more optionalsubstituents, only sterically practical and/or synthetically feasiblecompounds are meant to be included. “Optionally substituted” refers toall subsequent modifiers in a term or series of chemical groups. Forexample, in the term “optionally substituted arylalkyl, the “alkyl”portion and the “aryl” portion of the molecule may or may not besubstituted, and for the series “optionally substituted alkyl,cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, andheteroaryl groups, independently of the others, may or may not besubstituted.

“Protecting group” refers to a group of atoms that mask, reduce orprevent the reactivity of the functional group when attached to areactive functional group in a molecule. Typically, a protecting groupmay be selectively removed as desired during the course of a synthesis.Examples of protecting groups are known in the art (e.g., Wuts andGreene, “Greene's Protective Groups in Organic Synthesis,” 4^(th) Ed.,Wiley Interscience [2006], and Harrison et al., Compendium of SyntheticOrganic Methods, Vols. 1-8, John Wiley & Sons, NY [1971-1976].Functional groups that can have a protecting group include, but are notlimited to, hydroxy, amino, and carboxy groups. Representative aminoprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“SES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxyl protecting groupsinclude, but are not limited to, those where the hydroxyl group iseither acylated (e.g., methyl and ethyl esters, acetate or propionategroups or glycol esters) or alkylated such as benzyl and trityl ethers,as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers(e.g., TMS or TIPPS groups) and allyl ethers. Other protecting groupscan be found in the references noted herein.

“Leaving group” generally refers to any atom or moiety that is capableof being displaced by another atom or moiety in a chemical reaction.More specifically, a leaving group refers to an atom or moiety that isreadily displaced and substituted by a nucleophile (e.g., an amine, athiol, an alcohol, or cyanide). Such leaving groups are well known andinclude carboxylates, N-hydroxysuccinimide (“NHS”),N-hydroxybenzotriazole, a halogen (fluorine, chlorine, bromine, oriodine), and alkyloxy groups. Non-limiting characteristics and examplesof leaving groups are known in the art and described in variouschemistry texts.

Engineered Transaminase Polypeptides

The present disclosure provides engineered polypeptides havingtransaminase activity (also referred to herein as “engineeredtransaminase polypeptides”) useful for the selective transamination ofamino acceptor substrate compounds to produce chiral amine products,which, in some embodiments, can include compound (2). Accordingly, inone aspect, the present disclosure provides engineered polypeptideshaving transaminase activity which are capable of converting substratecompound (1) to product compound (2) as shown in Scheme 2. Further, thepresent disclosure provides polynucleotides encoding the engineeredpolypeptides, associated vectors and host cells comprising thepolynucleotides, methods for making the engineered polypeptides, andmethods for using the engineered polypeptides, including suitablereaction conditions.

The engineered polypeptides of the present disclosure are non-naturallyoccurring transaminases engineered to have improved enzyme properties(such as increased stereoselectivity) as compared to the wild-typetransaminase polypeptide of Vibrio fluvialis JS17 (GenBank Acc. No.AEA39183.1, GI: 327207066; SEQ ID NO:2), and also as compared to thereference engineered transaminase polypeptide of SEQ ID NO: 4, which wasused as the starting backbone sequence for the directed evolution of theengineered polypeptides of the present disclosure. The referenceengineered transaminase polypeptide of SEQ ID NO:4 has the following 11amino acid differences relative to the wild-type transaminase of Vibriofluvialis JS17 (SEQ ID NO:2): A9T, N45H, W57L, F86S, R88H, V153A, V177L,R211K, M294V, S324G, and T391A.

The engineered transaminase polypeptides of the present disclosure weregenerated by directed evolution of SEQ ID NO: 4 for efficient conversionof compound (1) to compound (2) under certain industrially relevantconditions and have one or more residue differences as compared to areference engineered transaminase polypeptide. These residue differencesare associated with improvements in various enzyme properties,particularly increased activity, increased stereoselectivity, increasedstability, and tolerance of increased substrate and/or productconcentration (e.g., decreased product inhibition). Accordingly, in someembodiments, the engineered polypeptides having transaminase activityare capable of converting the substrate compound (1) to compound (2)with an activity that is increased at least about 1.2 fold, 1.5 fold, 2fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50fold, 100 fold, or more relative to the activity of a referencepolypeptide (e.g., SEQ ID NO:2, 4, 8, 366, and/or 650), under suitablereaction conditions. In some embodiments, the engineered polypeptideshaving transaminase activity are capable of converting the substrate ofcompound (1) to compound (2) with a percent conversion of at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99%, in a reaction time of about 48 h, about 36 h, about 24 h, oreven a shorter length of time, under suitable reaction conditions. Insome embodiments, the engineered polypeptides having transaminaseactivity are capable of converting compound (1) to compound (2) indiastereomeric excess of at least 90%, 95%, 97%, 98%, 99%, or greater,under suitable reaction conditions.

The present disclosure provides numerous exemplary engineeredtransaminase polypeptides comprising amino acid sequences of theeven-numbered sequence identifiers SEQ ID NO: 6-936. These exemplaryengineered transaminase polypeptides comprise amino acid sequences thatinclude one or more of the following residue differences associated withtheir improved properties for conversion of compound (1) to compound (2)as compared to a reference sequence (e.g., SEQ ID NO: 2, 4, 8, 366,and/or 650).

In some cases, the exemplary engineered polypeptides have an amino acidsequence that further comprises one or more residue differences ascompared to a reference sequence (e.g., SEQ ID NO: 2, 4, 8, 366, and/or650). In some cases, the exemplary engineered polypeptides have an aminoacid sequence that further comprises one or more residue differences ascompared to a reference sequence (e.g., SEQ ID NO: 2, 4, 8, 366, and/or650).

In some embodiments, the engineered polypeptide comprises an amino acidsequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical to areference sequence selected from SEQ ID NO: 2, 4, 8, 366, and/or 650,where the polypeptide has transaminase activity and one or more of theimproved properties as described herein, for example the ability toconvert compound (1) to product compound (2) with increased activitycompared to a reference sequence (e.g., the polypeptide of SEQ ID NO: 2,4, 8, 366, and/or 650). In some embodiments, the reference sequence isSEQ ID NO: 2. In some embodiments, the reference sequence is SEQ ID NO:4. In some embodiments, the reference sequence is SEQ ID NO: 8. In someembodiments, the reference sequence is SEQ ID NO: 366. In someembodiments, the reference sequence is SEQ ID NO: 650.

In some embodiments, the engineered transaminase polypeptide comprisingan amino acid sequence has one or more amino acid residue differences ascompared to SEQ ID NO: 2, 4, 8, 366, and/or 650. In some embodiments,the present disclosure provides an engineered polypeptide havingtransaminase activity comprising an amino acid sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to reference sequence of SEQ ID NO:2, 4, 8, 366, and/or 650 and (a) at least one amino acid residuedifference selected from those substitutions provided herein (See e.g.,Tables 2-1, 2-2, 3-1, 3-2, 4-1, 4-2, 5-1, and/or 5-2).

In some embodiments, the present disclosure provides an engineeredtransaminase polypeptide comprising an amino acid sequence that has oneor more amino acid residue differences as compared to SEQ ID NO: 4selected 18, 20, 21, 21/23/56/146, 21/23/56/146/432, 21/23/146/417,21/23/395/417/432, 21/53/56, 21/53/417, 21/56/395, 21/417/432, 23,23/53, 23/53/56, 23/53/56/146/395, 23/53/395, 23/53/417, 23/53/432,23/56, 23/56/395, 23/56/395/417, 23/395/417, 23/417, 23/417/432, 53,53/56, 53/146/417, 53/395, 56, 56/74/241/286/314/316/323,56/86/163/314/316/383/414/416/422, 56/86/286/314/414/416,56/86/314/316/323/394/414/422, 56/146/417, 56/146/432, 56/147, 56/163,56/163/286/316/323/383/394, 56/286/314/316/323/422, 56/286/383, 56/323,56/323/383, 56/323/383/394, 56/383, 56/395, 74/81/286/316/323/383,74/85/86/163/286/316/323/394, 74/85/314/316/414/416, 74/86/163/316,74/86/316/323/383/394, 74/88/286/316/323/383, 74/88/323/383,74/163/286/316/383/394/416, 74/163/314/316, 74/163/314/316/323/394,74/163/314/323/383/414/416, 74/286, 74/286/316/323, 74/286/394/416,74/314/323/383/394/414, 74/316/323/394, 85/86/88/163/323/383/394,85/86/163/314/323/394/414, 85/286, 85/286/323, 86,86/88/163/323/383/414/422, 86/383/394, 88, 88/163/286/383,88/286/316/323, 88/286/316/323/383/414/416, 88/316/323, 146,146/147/395/417, 146/395, 146/395/417, 146/417, 147/395/417/432,147/417, 149, 157, 163, 163/222/286/316/323/383/394, 163/286,163/286/314/316/323/414/416, 163/286/314/323/394,163/286/316/323/394/416, 163/286/414, 163/314/316/394, 163/314/323/394,163/314/383, 163/314/414, 163/316/323, 163/323, 163/383, 164, 199/417,259, 260, 284, 286, 286/314/323/383, 286/314/394,286/316/323/383/414/416, 286/316/383/394, 286/316/394/414/416, 286/323,286/323/383/414, 286/323/416, 286/383, 286/416, 314/316, 314/316/323,314/316/323/383/422, 314/316/323/394, 314/316/394, 314/323/383/394,314/383, 314/383/414/422, 315, 316, 316/323/383/394,316/323/394/414/416, 316/414/422, 323, 323/383, 323/383/394/414/416,323/394, 383, 395, 395/417, 395/417/432, 400, 401, 403, 404, 405, 406,408, 415, 417, 417/432, 420, and 422, wherein the positions are numberedwith reference to SEQ ID NO: 4. In some embodiments, the amino aciddifferences comprise the substitution(s) 18A, 20C, 21H,21P/23S/56C/146H, 21P/23S/56C/146H/432V, 21P/23S/146H/417V,21P/23S/395D/417S/432V, 21P/53C/56C, 21P/53C/417S, 21P/56C/395D,21P/417S/432V, 21R, 23A, 23R, 23S/53C, 23S/53C/56C,23S/53C/56C/146H/395D, 23S/53C/395D, 23S/53C/417S, 23S/53C/432V,23S/56C, 23S/56C/395D, 23S/56C/395D/417V, 23S/395D/417S, 23S/417S,23S/417V, 23S/417V/432V, 53C, 53C/56C, 53C/146H/417S, 53C/395D, 56A,56A/74T/241V/286S/314R/316W/323T,56A/86A/163F/314R/316W/383V/414V/416A/422A, 56A/86A/286S/314R/414V/416A,56A/86A/314R/316W/323T/394G/414V/422A, 56A/163F,56A/163F/286S/316W/323T/383V/394G, 56A/286S/314R/316W/323T/422A,56A/286S/383V, 56A/323T, 56A/323T/383V, 56A/323T/383V/394G, 56A/383V,56C, 56C/146H/417V, 56C/146H/432V, 56C/147R, 56C/395D, 56T, 56V,74T/81S/286S/316W/323T/383V, 74T/85V/86A/163F/286S/316W/323T/394G,74T/85V/314R/316W/414V/416A, 74T/86A/163F/316W,74T/86A/316W/323T/383V/394G, 74T/88R/286S/316W/323T/383V,74T/88R/323T/383V, 74T/163F/286S/316W/383V/394G/416A,74T/163F/314R/316W, 74T/163F/314R/316W/323T/394G,74T/163F/314R/323T/383V/414V/416A, 74T/286S, 74T/286S/316W/323T,74T/286S/394G/416A, 74T/314R/323T/383V/394G/414V, 74T/316W/323T/394G,85V/86A/88R/163F/323T/383V/394G, 85V/86A/163F/314R/323T/394G/414V,85V/286S, 85V/286S/323T, 86A/88R/163F/323T/383V/414V/422A,86A/383V/394G, 86G, 88R/163F/286S/383V, 88R/286S/316W/323T,88R/286S/316W/323T/383V/414V/416A, 88R/316W/323T, 88S, 88T, 146H,146H/147R/395D/417S, 146H/395D, 146H/395D/417S, 146H/417S, 146H/417V,147R/395D/417S/432V, 147R/417S, 149S, 157A, 163F,163F/222V/286S/316W/323T/383V/394G, 163F/286S,163F/286S/314R/316W/323T/414V/416A, 163F/286S/314R/323T/394G,163F/286S/316W/323T/394G/416A, 163F/286S/414V, 163F/314R/316W/394G,163F/314R/323T/394G, 163F/314R/383V, 163F/314R/414V, 163F/316W/323T,163F/323T, 163F/383V, 163L, 163M, 164A, 164D, 164Q, 164S, 199V/417S,259V, 260T, 284A, 286S, 286S/314R/323T/383V, 286S/314R/394G,286S/316W/323T/383V/414V/416A, 286S/316W/383V/394G,286S/316W/394G/414V/416A, 286S/323T, 286S/323T/383V/414V,286S/323T/416A, 286S/383V, 286S/416A, 314R/316W, 314R/316W/323T,314R/316W/323T/383V/422A, 314R/316W/323T/394G, 314R/316W/394G,314R/323T/383V/394G, 314R/383V, 314R/383V/414V/422A, 315G, 315R, 316A,316F, 316G, 316H, 316L, 316N, 316R, 316V, 316W/323T/383V/394G,316W/323T/394G/414V/416A, 316W/414V/422A, 323C, 323S, 323T, 323T/383V,323T/383V/394G/414V/416A, 323T/394G, 383V, 395D, 395D/417S,395D/417S/432V, 395D/417V, 400D, 401A, 401K, 401S, 403V, 404S, 405H,405W, 406S, 408F, 408L, 408W, 415G, 415W, 417A, 417S, 417S/432V, 417V,417V/432V, 420G, 422L, and 422T, and 424R, wherein the positions arenumbered with reference to SEQ ID NO: 4. In some additional embodiments,the amino acid differences comprise the substitution(s) G18A, T20C,D21H, D21P/P23S/L56C/R146H, D21P/P23S/L56C/R146H/A432V,D21P/P23S/R146H/L417V, D21P/P23S/G395D/L417S/A432V, D21P/N53C/L56C,D21P/N53C/L417S, D21P/L56C/G395D, D21P/L417S/A432V, D21R, P23A, P23R,P23S/N53C, P23S/N53C/L56C, P23S/N53C/L56C/R146H/G395D, P23S/N53C/G395D,P23S/N53C/L417S, P23S/N53C/A432V, P23S/L56C, P23S/L56C/G395D,P23S/L56C/G395D/L417V, P23S/G395D/L417S, P23S/L417S, P23S/L417V,P23S/L417V/A432V, N53C, N53C/L56C, N53C/R146H/L417S, N53C/G395D, L56A,L56A/A74T/A241V/N286S/I314R/E316W/A323T,L56A/S86A/K163F/I314R/E316W/A383V/C414V/P416A/V422A,L56A/S86A/N286S/I314R/C414V/P416A,L56A/S86A/I314R/E316W/A323T/D394G/C414V/V422A, L56A/K163F,L56A/K163F/N286S/E316W/A323T/A383V/D394G,L56A/N286S/I314R/E316W/A323T/V422A, L56A/N286S/A383V, L56A/A323T,L56A/A323T/A383V, L56A/A323T/A383V/D394G, L56A/A383V, L56C,L56C/R146H/L417V, L56C/R146H/A432V, L56C/W147R, L56C/G395D, L56T, L56V,A74T/G81S/N286S/E316W/A323T/A383V,A74T/F85V/S86A/K163F/N286S/E316W/A323T/D394G,A74T/F85V/I314R/E316W/C414V/P416A, A74T/S86A/K163F/E316W,A74T/S86A/E316W/A323T/A383V/D394G, A74T/H88R/N286S/E316W/A323T/A383V,A74T/H88R/A323T/A383V, A74T/K163F/N286S/E316W/A383V/D394G/P416A,A74T/K163F/I314R/E316W, A74T/K163F/I314R/E316W/A323T/D394G,A74T/K163F/I314R/A323T/A383V/C414V/P416A, A74T/N286S,A74T/N286S/E316W/A323T, A74T/N286S/D394G/P416A,A74T/I314R/A323T/A383V/D394G/C414V, A74T/E316W/A323T/D394G,F85V/S86A/H88R/K163F/A323T/A383V/D394G,F85V/S86A/K163F/I314R/A323T/D394G/C414V, F85V/N286S, F85V/N286S/A323T,S86A/H88R/K163F/A323T/A383V/C414V/V422A, S86A/A383V/D394G, S86G,H88R/K163F/N286S/A383V, H88R/N286S/E316W/A323T,H88R/N286S/E316W/A323T/A383V/C414V/P416A, H88R/E316W/A323T, H88S, H88T,R146H, R146H/W147R/G395D/L417S, R146H/G395D, R146H/G395D/L417S,R146H/L417S, R146H/L417V, W147R/G395D/L417S/A432V, W147R/L417S, A149S,S157A, K163F, K163F/A222V/N286S/E316W/A323T/A383V/D394G, K163F/N286S,K163F/N286S/I314R/E316W/A323T/C414V/P416A,K163F/N286S/I314R/A323T/D394G, K163F/N286S/E316W/A323T/D394G/P416A,K163F/N286S/C414V, K163F/I314R/E316W/D394G, K163F/I314R/A323T/D394G,K163F/I314R/A383V, K163F/I314R/C414V, K163F/E316W/A323T, K163F/A323T,K163F/A383V, K163L, K163M, P164A, P164D, P164Q, P164S, A199V/L417S,I259V, C260T, S284A, N286S, N286S/I314R/A323T/A383V, N286S/I314R/D394G,N286S/E316W/A323T/A383V/C414V/P416A, N286S/E316W/A383V/D394G,N286S/E316W/D394G/C414V/P416A, N286S/A323T, N286S/A323T/A383V/C414V,N286S/A323T/P416A, N286S/A383V, N286S/P416A, I314R/E316W,I314R/E316W/A323T, I314R/E316W/A323T/A383V/V422A,I314R/E316W/A323T/D394G, I314R/E316W/D394G, I314R/A323T/A383V/D394G,I314R/A383V, I314R/A383V/C414V/V422A, E315G, E315R, E316A, E316F, E316G,E316H, E316L, E316N, E316R, E316V, E316W/A323T/A383V/D394G,E316W/A323T/D394G/C414V/P416A, E316W/C414V/V422A, A323C, A323S, A323T,A323T/A383V, A323T/A383V/D394G/C414V/P416A, A323T/D394G, A383V, G395D,G395D/L417S, G395D/L417S/A432V, G395D/L417V, S400D, E401A, E401K, E401S,I403V, A404S, N405H, N405W, T406S, T408F, T408L, T408W, R415G, R415W,L417A, L417S, L417S/A432V, L417V, L417V/A432V, S420G, V422L, and V422T,wherein the positions are numbered with reference to SEQ ID NO: 4.

In some embodiments, the present disclosure provides an engineeredtransaminase polypeptide comprising an amino acid sequence that has oneor more amino acid residue differences as compared to SEQ ID NO: 4selected from 74/81/286/316/323/383, 163/286/314/316/323/414/416,163/286/314/323/394, 286/314/323/383, 286/316/323/383/414/416, 315, and408, wherein the positions are numbered with reference to SEQ ID NO: 4.In some embodiments, the amino acid difference(s) comprise thesubstitution(s) 74T/81S/286S/316W/323T/383V,163F/286S/314R/316W/323T/414V/416A, 163F/286S/314R/323T/394G,286S/314R/323T/383V, 286S/316W/323T/383V/414V/416A, 315G, and 408F,wherein the positions are numbered with reference to SEQ ID NO: 4. Insome additional embodiments, the amino acid difference(s) comprise thesubstitution(s) A74T/G81S/N286S/E316W/A323T/A383V,K163F/N286S/I314R/E316W/A323T/C414V/P416A,K163F/N286S/I314R/A323T/D394G, N286S/I314R/A323T/A383V,N286S/E316W/A323T/A383V/C414V/P416A, E315G, and T408F, wherein thepositions are numbered with reference to SEQ ID NO: 4.

In some embodiments, the present disclosure provides an engineeredtransaminase polypeptide comprising an amino acid sequence that has oneor more amino acid residue differences as compared to SEQ ID NO: 8selected from 5, 18/23/149/260/383/395/401/416, 18/23/149/383,18/163/164, 21, 21/163/315/316, 21/163/323/408, 21/408,23/56/86/149/163/164/383/401/416, 23/86, 23/149/260, 23/149/284/383/395,23/163/164/383, 23/163/164/401/416, 24, 42, 42/110, 42/187/272,42/187/324/363/366, 42/187/353, 42/272/291, 42/272/291/363,42/272/324/363/366, 42/272/363/410, 42/272/410, 42/291/313/363/410,42/291/363, 42/291/363/366, 42/353, 42/363, 46, 66, 77,86/149/163/164/383/395/401, 86/149/395, 86/163/164/260/383, 86/383, 107,110, 110/187, 110/187/253/410, 134, 138, 149/164/260/383/395/401,149/260/383, 149/416, 163/259/323/408, 163/259/408, 163/315/316,164/260/401, 164/316/383/401, 167, 186, 187, 187/253/363/366,187/272/324/363/410, 187/272/363, 187/272/363/366/410, 187/291, 189,191, 195, 199, 203, 210, 211, 248, 259/307, 260/395/401, 272, 272/353,272/363/366, 272/410, 277, 291, 305, 309, 315, 342, 343, 351, 354, 358,361, 362, 363, 363/366, 365, 367, 383, 383/401, 383/416/422, 385, 388,389, 392, 395, 396, 401, 404, 405, 408, 410, 416, 417, 439, 443, 447,450, and 451, wherein the positions are numbered with reference to SEQID NO: 8. In some embodiments, the amino acid difference(s) comprise thesubstitution(s) 5E, 5G, 18A/23R/149S/260T/383V/395D/401S/416P,18A/23R/149S/383V, 18A/163M/164Q, 21H, 21H/163L/315G/316F,21H/163L/323C/408F, 21H/408F, 23R/56C/86G/149S/163M/164D/383V/401S/416P,23R/86G, 23R/149S/260T, 23R/149S/284A/383V/395D, 23R/163M/164Q/383V,23R/163M/164S/401S/416P, 24K, 24R, 42F, 42F/110K, 42F/187E/272E,42F/187E/324S/363L/366H, 42F/187E/353T, 42F/272E/291Y,42F/272E/291Y/363L, 42F/272E/324S/363L/366H, 42F/272E/363L/410H,42F/272E/410H, 42F/291Y/313V/363L/410H, 42F/291Y/363L,42F/291Y/363L/366H, 42F/353T, 42F/363L, 46S, 66A, 77M,86G/149S/163M/164S/383V/395D/401S, 86G/149S/395D,86G/163M/164S/260T/383V, 86G/383V, 107L, 107S, 107Y, 110K, 110K/187E,110K/187E/253L/410H, 134V, 138R, 149S/164S/260T/383V/395D/401A,149S/260T/383V, 149S/416P, 163L/259V/323C/408F, 163L/259V/408F,163L/315G/316F, 164D/316H/383V/401S, 164S/260T/401S, 167N, 186Q, 187E,187E/253L/363L/366H, 187E/272E/324S/363L/410H, 187E/272E/363L,187E/272E/363L/366H/410H, 187E/291Y, 189F, 189S, 189V, 189W, 191D, 191F,195W, 199Q, 203L, 210A, 210L, 210M, 210V, 210Y, 211R, 248G, 259V/307M,260T/395D/401S, 272E, 272E/353T, 272E/363L/366H, 272E/410H, 277S, 291Y,305E, 309A, 309F, 309R, 315G, 342T, 343G, 351L, 354S, 358L, 361R, 362Q,362V, 363L, 363L/366H, 365L, 365Q, 365R, 365S, 367T, 383V, 383V/401A,383V/416P/422T, 385L, 385T, 388D, 388L, 388P, 389D, 392A, 392L, 395R,396P, 396Y, 401Q, 401S, 404M, 405W, 408A, 408E, 408W, 410H, 416P, 417S,439L, 439S, 443L, 443S, 447S, 447T, 450D, and 451S, wherein thepositions are numbered with reference to SEQ ID NO: 8. In someadditional embodiments, the amino acid difference(s) comprise thesubstitution(s) Q5E, Q5G, G18A/P23R/A149S/C260T/A383V/G395D/E401S/A416P,G18A/P23R/A149S/A383V, G18A/F163M/P164Q, D21H, D21H/F163L/E315G/W316F,D21H/F163L/T323C/T408F, D21H/T408F,P23R/L56C/S86G/A149S/F163M/P164D/A383V/E401S/A416P, P23R/S86G,P23R/A149S/C260T, P23R/A149S/S284A/A383V/G395D, P23R/F163M/P164Q/A383V,P23R/F163M/P164S/E401S/A416P, S24K, S24R, V42F, V42F/R110K,V42F/Y187E/V272E, V42F/Y187E/G324S/I363L/R366H, V42F/Y187E/A353T,V42F/V272E/F291Y, V42F/V272E/F291Y/I363L, V42F/V272E/G324S/I363L/R366H,V42F/V272E/I363L/L410H, V42F/V272E/L410H, V42F/F291Y/A313V/I363L/L410H,V42F/F291Y/I363L, V42F/F291Y/I363L/R366H, V42F/A353T, V42F/I363L, G46S,K66A, E77M, S86G/A149S/F163M/P164S/A383V/G395D/E401S, S86G/A149S/G395D,S86G/F163M/P164S/C260T/A383V, S86G/A383V, D107L, D107S, D107Y, R110K,R110K/Y187E, R110K/Y187E/V253L/L410H, A134V, P138R,A149S/P164S/C260T/A383V/G395D/E401A, A149S/C260T/A383V, A149S/A416P,F163L/I259V/T323C/T408F, F163L/I259V/T408F, F163L/E315G/W316F,P164D/W316H/A383V/E401S, P164S/C260T/E401S, S167N, R186Q, Y187E,Y187E/V253L/I363L/R366H, Y187E/V272E/G324S/I363L/L410H,Y187E/V272E/I363L, Y187E/V272E/I363L/R366H/L410H, Y187E/F291Y, E189F,E189S, E189V, E189W, G191D, G191F, E195W, A199Q, R203L, Q210A, Q210L,Q210M, Q210V, Q210Y, K211R, K248G, I259V/L307M, C260T/G395D/E401S,V272E, V272E/A353T, V272E/I363L/R366H, V272E/L410H, T277S, F291Y, K305E,T309A, T309F, T309R, E315G, N342T, E343G, R351L, P354S, E358L, K361R,H362Q, H362V, I363L, I363L/R366H, E365L, E365Q, E365R, E365S, P367T,A383V, A383V/E401A, A383V/A416P/V422T, K385L, K385T, A388D, A388L,A388P, S389D, P392A, P392L, G395R, N396P, N396Y, E401Q, E401S, A404M,N405W, T408A, T408E, T408W, L410H, A416P, L417S, D439L, D439S, K443L,K443S, K447S, K447T, A450D, and E451S, wherein the positions arenumbered with reference to SEQ ID NO: 8.

In some embodiments, the present disclosure provides an engineeredtransaminase polypeptide comprising an amino acid sequence that has oneor more amino acid residue differences as compared to SEQ ID NO: 8selected from 18/23/149/383, 21/163/323/408, 272, 291, and 383, whereinthe positions are numbered with reference to SEQ ID NO: 8. In someembodiments, the amino acid difference(s) comprise the substitution(s)18A/23R/149S/383V, 21H/163L/323C/408F, 272E, 291Y, and 383V, wherein thepositions are numbered with reference to SEQ ID NO: 8. In someadditional embodiments, the amino acid difference(s) comprise thesubstitution(s) G18A/P23R/A149S/A383V, D21H/F163L/T323C/T408F, V272E,F291Y, and A383V, wherein the positions are numbered with reference toSEQ ID NO: 8.

In some embodiments, the present disclosure provides an engineeredtransaminase polypeptide comprising an amino acid sequence that has oneor more amino acid residue differences as compared to SEQ ID NO: 366selected from 24, 24/42/66/291, 24/42/291/362,24/66/163/191/362/383/388, 24/66/191/199/260/291/351, 24/66/191/199/291,24/66/191/260/408, 24/66/260/291/383/388/408, 24/66/291/342/383,24/66/291/365, 24/66/342/365/388/408, 24/77/291,24/107/163/191/291/351/383/388, 24/107/291/351/365/388, 24/163/351/383,24/191/291/365, 24/199/260/351/362/383, 24/199/260/362/383/388,24/260/362/383/388, 24/291, 24/291/342/351/383, 24/291/362/388,24/291/408, 24/383/388, 24/388, 25, 28, 33, 42/191/408, 42/199/291/383,42/291/351/362/365/383/388, 42/291/351/362/383/408, 42/291/383/388,66/82/291/383, 66/163/191/365/383, 66/199/351/383, 66/291,66/291/362/365/383, 66/291/383/388, 66/383, 77/291, 77/383/388, 86,107/191/199/365/383/388, 107/191/291/383, 148, 153,163/291/362/365/383/388, 163/291/383/388, 163/383, 191/199/365/383/388,191/260/388, 191/291, 191/291/342/362/365, 191/351/383/388, 199/260/383,199/291, 260, 260/291/365/383/408, 260/365/383, 291, 291/351/383/388,291/351/383/388/408, 291/362/365, 291/365/388, 291/383, 314, 315, 316,319, 342/362, 351/383/388, 362, 362/388, 383, 383/388, 396, 397, 405,406, 413, 419, and 423, wherein the positions are numbered withreference to SEQ ID NO: 366. In some embodiments, the amino aciddifference(s) comprise the substitution(s) 24E, 24K, 24K/42F/66A/291Y,24K/42F/291Y/362Q, 24K/66A/163M/191D/362Q/383V/388D,24K/66A/191D/199Q/260T/291Y/351L, 24K/66A/191D/199Q/291Y,24K/66A/191D/260T/408A, 24K/66A/260T/291Y/383V/388D/408A,24K/66A/291Y/342T/383V, 24K/66A/291Y/365S, 24K/66A/342T/365S/388D/408E,24K/77M/291Y, 24K/107L/163M/191D/291Y/351L/383V/388D,24K/107L/291Y/351L/365S/388D, 24K/163M/351L/383V, 24K/191D/291Y/365S,24K/199Q/260T/351L/362Q/383V, 24K/199Q/260T/362Q/383V/388D,24K/260T/362Q/383V/388D, 24K/291Y, 24K/291Y/342T/351L/383V,24K/291Y/362Q/388D, 24K/291Y/408A, 24K/383V/388D, 24K/388D, 25H, 25V,28S, 28T, 33T, 42F/191D/408E, 42F/199Q/291Y/383V,42F/291Y/351L/362Q/365S/383V/388D, 42F/291Y/351L/362Q/383V/408A,42F/291Y/383V/388D, 66A/82H/291Y/383V, 66A/163M/191D/365S/383V,66A/199Q/351L/383V, 66A/291Y, 66A/291Y/362Q/365S/383V,66A/291Y/383V/388D, 66A/383V, 77M/291Y, 77M/383V/388D, 86T,107L/191D/199Q/365S/383V/388D, 107L/191D/291Y/383V, 148G, 153S,163M/291Y/362Q/365S/383V/388D, 163M/291Y/383V/388D, 163M/383V,191D/199Q/365S/383V/388D, 191D/260T/388D, 191D/291Y,191D/291Y/342T/362Q/365S, 191D/351L/383V/388D, 199Q/260T/383V,199Q/291Y, 260T, 260T/291Y/365S/383V/408A, 260T/365S/383V, 291Y,291Y/351L/383V/388D, 291Y/351L/383V/388D/408A, 291Y/362Q/365S,291Y/365S/388D, 291Y/383V, 314K, 315S, 316V, 319S, 342T/362Q,351L/383V/388D, 362Q, 362Q/388D, 383V, 383V/388D, 396R, 397M, 405A,406H, 413L, 419S, and 423V, wherein the positions are numbered withreference to SEQ ID NO: 366. In some additional embodiments, the aminoacid difference(s) comprise the substitution(s) S24E, S24K,S24K/V42F/K66A/F291Y, S24K/V42F/F291Y/H362Q,S24K/K66A/L163M/G191D/H362Q/A383V/A388D,S24K/K66A/G191D/A199Q/C260T/F291Y/R351L, S24K/K66A/G191D/A199Q/F291Y,S24K/K66A/G191D/C260T/F408A, S24K/K66A/C260T/F291Y/A383V/A388D/F408A,S24K/K66A/F291Y/N342T/A383V, S24K/K66A/F291Y/E365S,S24K/K66A/N342T/E365S/A388D/F408E, S24K/E77M/F291Y,S24K/D107L/L163M/G191D/F291Y/R351L/A383V/A388D,S24K/D107L/F291Y/R351L/E365S/A388D, S24K/L163M/R351L/A383V,S24K/G191D/F291Y/E365S, S24K/A199Q/C260T/R351L/H362Q/A383V,S24K/A199Q/C260T/H362Q/A383V/A388D, S24K/C260T/H362Q/A383V/A388D,S24K/F291Y, S24K/F291Y/N342T/R351L/A383V, S24K/F291Y/H362Q/A388D,S24K/F291Y/F408A, S24K/A383V/A388D, S24K/A388D, L25H, L25V, R28S, R28T,V33T, V42F/G191D/F408E, V42F/A199Q/F291Y/A383V,V42F/F291Y/R351L/H362Q/E365S/A383V/A388D,V42F/F291Y/R351L/H362Q/A383V/F408A, V42F/F291Y/A383V/A388D,K66A/Y82H/F291Y/A383V, K66A/L163M/G191D/E365S/A383V,K66A/A199Q/R351L/A383V, K66A/F291Y, K66A/F291Y/H362Q/E365S/A383V,K66A/F291Y/A383V/A388D, K66A/A383V, E77M/F291Y, E77M/A383V/A388D, S86T,D107L/G191D/A199Q/E365S/A383V/A388D, D107L/G191D/F291Y/A383V, N148G,A153S, L163M/F291Y/H362Q/E365S/A383V/A388D, L163M/F291Y/A383V/A388D,L163M/A383V, G191D/A199Q/E365S/A383V/A388D, G191D/C260T/A388D,G191D/F291Y, G191D/F291Y/N342T/H362Q/E365S, G191D/R351L/A383V/A388D,A199Q/C260T/A383V, A199Q/F291Y, C260T, C260T/F291Y/E365S/A383V/F408A,C260T/E365S/A383V, F291Y, F291Y/R351L/A383V/A388D,F291Y/R351L/A383V/A388D/F408A, F291Y/H362Q/E365S, F291Y/E365S/A388D,F291Y/A383V, R314K, E315S, W316V, H319S, N342T/H362Q, R351L/A383V/A388D,H362Q, H362Q/A388D, A383V, A383V/A388D, N396R, L397M, N405A, T406H,I413L, Q419S, and L423V, wherein the positions are numbered withreference to SEQ ID NO: 366.

In some embodiments, the present disclosure provides an engineeredtransaminase polypeptide comprising an amino acid sequence that has oneor more amino acid residue differences as compared to SEQ ID NO: 366selected from 24/66/191/199/291, 24/66/291/365, 163/291/362/365/383/388,163/291/383/388, 191/291/342/362/365, 291, and 291/383, wherein thepositions are numbered with reference to SEQ ID NO: 366. In someembodiments, the amino acid difference(s) comprise the substitution(s)24K/66A/191D/199Q/291Y, 24K/66A/291Y/365S,163M/291Y/362Q/365S/383V/388D, 163M/291Y/383V/388D,191D/291Y/342T/362Q/365S, 291Y, and 291Y/383V, wherein the positions arenumbered with reference to SEQ ID NO: 366. In some additionalembodiments, the amino acid difference(s) comprise the substitution(s)S24K/K66A/G191D/A199Q/F291Y, S24K/K66A/F291Y/E365S,L163M/F291Y/H362Q/E365S/A383V/A388D, L163M/F291Y/A383V/A388D,G191D/F291Y/N342T/H362Q/E365S, F291Y, and F291Y/A383V, wherein thepositions are numbered with reference to SEQ ID NO: 366.

In some embodiments, the present disclosure provides an engineeredtransaminase polypeptide comprising an amino acid sequence that has oneor more amino acid residue differences as compared to SEQ ID NO: 650selected from 10, 13, 13/24/108/163, 13/24/108/163/311,13/24/133/199/311, 13/24/163, 13/24/199/311, 13/108, 13/108/199,13/108/311, 13/199, 13/311, 14, 14/24/108, 14/24/108/133, 14/24/108/199,14/24/199, 14/108, 14/108/133/311, 14/108/311, 14/311, 24, 24/163,24/163/199, 35, 72, 73, 78, 95, 101, 108, 108/199, 114, 154, 163, 169,175/316, 199, 199/311, 226, 293, 311, 316, 382, 383, and 386, whereinthe positions are numbered with reference to SEQ ID NO: 650. In someembodiments, the amino acid difference(s) comprise the substitution(s)10E, 13A, 13A/24E/108R/163L/311S, 13A/24E/133R/199Q/311S, 13A/24E/163L,13A/24K/108R/163L, 13A/24K/199Q/311S, 13A/108R, 13A/108R/199Q,13A/108R/311S, 13A/199Q, 13A/311S, 14A, 14G, 14H, 14H/24E/108R/133R,14H/24K/108R, 14H/24K/108R/199Q, 14H/24K/199Q, 14H/108R,14H/108R/133R/311S, 14H/108R/311S, 14H/311S, 24E, 24E/163L,24E/163L/199Q, 35E, 72G, 73R, 73S, 78A, 95I, 101L, 108R, 108R/199Q,114A, 154S, 163H, 163S, 163V, 169C, 169V, 175D/316F, 199Q, 199Q/311S,226Q, 293A, 311K, 316D, 316E, 316F, 316G, 316H, 316I, 316L, 316N, 316S,316V, 316Y, 382D, 383L, and 386A, wherein the positions are numberedwith reference to SEQ ID NO: 650. In some additional embodiments, theamino acid difference(s) comprise the substitution(s) R10E, T13A,T13A/S24E/S108R/M163L/I311S, T13A/S24E/A133R/A199Q/I311S,T13A/S24E/M163L, T13A/S24K/S108R/M163L, T13A/S24K/A199Q/I311S,T13A/S108R, T13A/S108R/A199Q, T13A/S108R/I311S, T13A/A199Q, T13A/I311S,Yl4A, Yl4G, Yl4H, Y14H/S24E/S108R/A133R, Y14H/S24K/S108R,Y14H/S24K/S108R/A199Q, Y14H/S24K/A199Q, Y14H/S108R,Y14H/S108R/A133R/I311S, Y14H/S108R/I311S, Y14H/I311S, S24E, S24E/M163L,S24E/M163L/A199Q, H35E, A72G, K73R, K73S, R78A, M95I, V101L, S108R,S108R/A199Q, T114A, T154S, M163H, M163S, M163V, F169C, F169V,G175D/W316F, A199Q, A199Q/1311S, M226Q, P293A, I311K, W316D, W316E,W316F, W316G, W316H, W316I, W316L, W316N, W316S, W316V, W316Y, E382D,V383L, and D386A, wherein the positions are numbered with reference toSEQ ID NO: 650.

In some embodiments, the present disclosure provides an engineeredtransaminase polypeptide comprising an amino acid sequence that has oneor more amino acid residue differences as compared to SEQ ID NO: 650selected from 14/108/133/311, 24/163/199, 72, 78, 316, and 383, whereinthe positions are numbered with reference to SEQ ID NO: 650. In someembodiments, the amino acid difference(s) comprise the substitution(s)14H/108R/133R/311S, 24E/163L/199Q, 72G, 78A, 316L, 316N, 316S, and 383L,wherein the positions are numbered with reference to SEQ ID NO: 650. Insome additional embodiments, the amino acid difference(s) comprise thesubstitution(s) Y14H/S108R/A133R/I311S, S24E/M163L/A199Q, A72G, R78A,W316L, W316N, W316S, and V383L, wherein the positions are numbered withreference to SEQ ID NO: 650.

In some embodiments, the engineered polypeptides having transaminaseactivity are capable of converting compound (1) to compound (2) withincreased tolerance for the presence of the substrate relative to thesubstrate tolerance of a reference polypeptide (e.g., SEQ ID NO: 2, 4,8, 366, and/or 650), under suitable reaction conditions. Accordingly, insome embodiments the engineered polypeptides are capable of convertingthe substrate of compound (1) to compound (2) in the presence of asubstrate loading concentration of at least about 1 g/L, 5 g/L, 10 g/L,20 g/L, about 30 g/L, about 40 g/L, about 50 g/L, about 70 g/L, about 75g/L, about 100 g/L, with a percent conversion of at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%, in areaction time of about 72h, about 48h, about 36h, about 24 h, or evenshorter length of time, under suitable reaction conditions.

Some suitable reaction conditions under which the above-describedimproved properties of the engineered polypeptides can be determinedwith respect concentrations or amounts of polypeptide, substrate, aminedonor, cofactor, buffer, co-solvent, pH, and/or conditions includingtemperature and reaction time are provided herein. In some embodiments,the suitable reaction conditions comprise the HTP, SFP, or DSP assayconditions described below and in the Examples.

As will be apparent to the skilled artisan, the foregoing residuepositions and the specific amino acid residues for each residue positioncan be used individually or in various combinations to synthesizetransaminase polypeptides having desired improved properties, including,among others, enzyme activity, substrate/product preference,stereoselectivity, substrate/product tolerance, and stability undervarious conditions, such as increased temperature, solvent, and/or pH.

In some embodiments, the present disclosure also provides engineeredtransaminase polypeptides that comprise a fragment of any of theengineered transaminase polypeptides described herein that retains thefunctional transaminase activity and/or improved property of thatengineered transaminase polypeptide. Accordingly, in some embodiments,the present disclosure provides a polypeptide fragment havingtransaminase activity (e.g., capable of converting compound (1) tocompound (2) under suitable reaction conditions), wherein the fragmentcomprises at least about 80%, 90%, 95%, 98%, or 99% of a full-lengthamino acid sequence of an engineered polypeptide of the presentdisclosure, such as an exemplary engineered polypeptide of having theeven-numbered sequence identifiers of SEQ ID NO: 6-936.

In some embodiments, the engineered transaminase polypeptide of thedisclosure comprises an amino acid sequence comprising a deletion ascompared to any one of the engineered transaminase polypeptide sequencesdescribed herein, such as the exemplary engineered polypeptide sequenceshaving the even-numbered sequence identifiers of SEQ ID NO: 6-936. Thus,for each and every embodiment of the engineered transaminasepolypeptides of the disclosure, the amino acid sequence can comprisedeletions of one or more amino acids, 2 or more amino acids, 3 or moreamino acids, 4 or more amino acids, 5 or more amino acids, 6 or moreamino acids, 8 or more amino acids, 10 or more amino acids, 15 or moreamino acids, or 20 or more amino acids, up to 10% of the total number ofamino acids, up to 10% of the total number of amino acids, up to 20% ofthe total number of amino acids, or up to 30% of the total number ofamino acids of the transaminase polypeptides, where the associatedfunctional activity and/or improved properties of the engineeredtransaminase described herein is maintained. In some embodiments, thedeletions can comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50,1-55, or 1-60 amino acid residues. In some embodiments, the number ofdeletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, 50, 55, or 60amino acid residues. In some embodiments, the deletions can comprisedeletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, 25 or 30 amino acid residues.

In some embodiments, the present disclosure provides an engineeredtransaminase polypeptide having an amino acid sequence comprising aninsertion as compared to any one of the engineered transaminasepolypeptide sequences described herein, such as the exemplary engineeredpolypeptide sequences having the even-numbered sequence identifiers ofSEQ ID NO:6-936. Thus, for each and every embodiment of the transaminasepolypeptides of the disclosure, the insertions can comprise one or moreamino acids, 2 or more amino acids, 3 or more amino acids, 4 or moreamino acids, 5 or more amino acids, 6 or more amino acids, 8 or moreamino acids, 10 or more amino acids, 15 or more amino acids, or 20 ormore amino acids, where the associated functional activity and/orimproved properties of the engineered transaminase described herein ismaintained. The insertions can be to amino or carboxy terminus, orinternal portions of the transaminase polypeptide.

In some embodiments, the polypeptides of the present disclosure are inthe form of fusion polypeptides in which the engineered polypeptides arefused to other polypeptides, such as, by way of example and notlimitation, antibody tags (e.g., myc epitope), purification sequences(e.g., His tags for binding to metals), and cell localization signals(e.g., secretion signals). Thus, the engineered polypeptides describedherein can be used with or without fusions to other polypeptides.

The engineered transaminase polypeptides described herein are notrestricted to the genetically encoded amino acids. Thus, in addition tothe genetically encoded amino acids, the polypeptides described hereinmay be comprised, either in whole or in part, of naturally-occurringand/or synthetic non-encoded amino acids. Certain commonly encounterednon-encoded amino acids of which the polypeptides described herein maybe comprised include, but are not limited to: the D-stereoisomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine(Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutamic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art. These amino acids may be in either the L- orD-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(S-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

In some embodiments, the engineered polypeptides can be provided on asolid support, such as a membrane, resin, solid carrier, or other solidphase material. A solid support can be composed of organic polymers suchas polystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of a solid support can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation.

In some embodiments, the engineered polypeptides having transaminaseactivity are bound or immobilized on the solid support such that theyretain their improved activity, enantioselectivity, stereoselectivity,and/or other improved properties relative to a reference polypeptide(e.g., SEQ ID NO: 2, 4, 8, 366, and/or 650). In such embodiments, theimmobilized polypeptides can facilitate the biocatalytic conversion ofthe substrate compound to the desired product, and after the reaction iscomplete are easily retained (e.g., by retaining beads on whichpolypeptide is immobilized) and then reused or recycled in subsequentreactions. Such immobilized enzyme processes allow for furtherefficiency and cost reduction. Accordingly, it is further contemplatedthat any of the methods of using the engineered transaminasepolypeptides of the present disclosure can be carried out using the sametransaminase polypeptides bound or immobilized on a solid support.

The engineered transaminase polypeptide can be bound non-covalently orcovalently. Various methods for conjugation and immobilization ofenzymes to solid supports (e.g., resins, membranes, beads, glass, etc.)are well known in the art. In particular, PCT publication WO2012/177527A1 discloses immobilized engineered transaminase polypeptides capable ofconverting compound (2) to compound (1), and methods of preparing theimmobilized polypeptides, in which the polypeptide is physicallyattached to a resin by either hydrophobic interactions or covalentbonds, and is stable in a solvent system that comprises at least up to100% organic solvent. Other methods for conjugation and immobilizationof enzymes to solid supports (e.g., resins, membranes, beads, glass,etc.) are well known in the art (See e.g., Yi et al., Proc. Biochem.,42: 895-898 [2007]; Martin et al., Appl. Microbiol. Biotechnol., 76:843-851 [2007]; Koszelewski et al., J. Mol. Cat. B: Enz., 63: 39-44[2010]; Truppo et al., Org. Proc. Res. Develop., published online:dx.doi.org/10.1021/op200157c; and Mateo et al., Biotechnol. Prog.,18:629-34 [2002], etc.).

Solid supports useful for immobilizing the engineered transaminasepolypeptides of the present disclosure include but are not limited tobeads or resins comprising polymethacrylate with epoxide functionalgroups, polymethacrylate with amino epoxide functional groups,styrene/DVB copolymer or polymethacrylate with octadecyl functionalgroups. Exemplary solid supports useful for immobilizing the engineeredtransaminases of the present disclosure include, but are not limited to,chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi), including thefollowing different types of SEPABEAD: EC-EP, EC-HFA/S, EXA252, EXE119and EXE120.

In some embodiments, the engineered transaminase polypeptides can beprovided in the form of an array in which the polypeptides are arrangedin positionally distinct locations. In some embodiments, thepositionally distinct locations are wells in a solid support such as a96-well plate. A plurality of supports can be configured on an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments. Such arrays can be used to test avariety of substrate compounds for conversion by the polypeptides.

In some embodiments, the engineered polypeptides described herein can beprovided in the form of kits. The polypeptides in the kits may bepresent individually or as a plurality of polypeptides. The kits canfurther include reagents for carrying out enzymatic reactions,substrates for assessing the activity of polypeptides, as well asreagents for detecting the products. The kits can also include reagentdispensers and instructions for use of the kits. In some embodiments,the kits of the present disclosure include arrays comprising a pluralityof different engineered transaminase polypeptides at differentaddressable position, wherein the different polypeptides are differentvariants of a reference sequence each having at least one differentimproved enzyme property. Such arrays comprising a plurality ofengineered polypeptides and methods of their use are known (See e.g.,WO2009/008908A2).

Polynucleotides, Control Sequences, Expression Vectors, and Host CellsUseful for Preparing Engineered Transaminase Polypeptides

In another aspect, the present disclosure provides polynucleotidesencoding the engineered polypeptides having transaminase activitydescribed herein. The polynucleotides may be operatively linked to oneor more heterologous regulatory sequences that control gene expressionto create a recombinant polynucleotide capable of expressing thepolypeptide. Expression constructs containing a heterologouspolynucleotide encoding the engineered transaminase can be introducedinto appropriate host cells to express the corresponding engineeredtransaminase polypeptide.

In some embodiments, the isolated polynucleotide encoding an improvedtransaminase polypeptide is manipulated in a variety of ways to providefor improved activity and/or expression of the polypeptide. Manipulationof the isolated polynucleotide prior to its insertion into a vector maybe desirable or necessary depending on the expression vector. Thetechniques for modifying polynucleotides and nucleic acid sequencesutilizing recombinant DNA methods are well known in the art.

Those of ordinary skill in the art understand that due to the degeneracyof the genetic code, a multitude of nucleotide sequences encodingvariant transaminase acylase polypeptides of the present disclosureexist. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the disclosure where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that “U” inan RNA sequence corresponds to “T” in a DNA sequence. The disclosurecontemplates and provides each and every possible variation of nucleicacid sequence encoding a polypeptide of the disclosure that could bemade by selecting combinations based on possible codon choices.

As indicated above, DNA sequence encoding a transaminase may also bedesigned for high codon usage bias codons (codons that are used athigher frequency in the protein coding regions than other codons thatcode for the same amino acid). The preferred codons may be determined inrelation to codon usage in a single gene, a set of genes of commonfunction or origin, highly expressed genes, the codon frequency in theaggregate protein coding regions of the whole organism, codon frequencyin the aggregate protein coding regions of related organisms, orcombinations thereof. A codon whose frequency increases with the levelof gene expression is typically an optimal codon for expression. Inparticular, a DNA sequence can be optimized for expression in aparticular host organism. A variety of methods are well-known in the artfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis (e.g., using cluster analysis orcorrespondence analysis,) and the effective number of codons used in agene. The data source for obtaining codon usage may rely on anyavailable nucleotide sequence capable of coding for a protein. Thesedata sets include nucleic acid sequences actually known to encodeexpressed proteins (e.g., complete protein coding sequences-CDS),expressed sequence tags (ESTs), or predicted coding regions of genomicsequences, as is well-known in the art. Polynucleotides encoding varianttransaminases can be prepared using any suitable methods known in theart. Typically, oligonucleotides are individually synthesized, thenjoined (e.g., by enzymatic or chemical ligation methods, orpolymerase-mediated methods) to form essentially any desired continuoussequence. In some embodiments, polynucleotides of the present disclosureare prepared by chemical synthesis using, any suitable methods known inthe art, including but not limited to automated synthetic methods. Forexample, in the phosphoramidite method, oligonucleotides are synthesized(e.g., in an automatic DNA synthesizer), purified, annealed, ligated andcloned in appropriate vectors. In some embodiments, double stranded DNAfragments are then obtained either by synthesizing the complementarystrand and annealing the strands together under appropriate conditions,or by adding the complementary strand using DNA polymerase with anappropriate primer sequence. There are numerous general and standardtexts that provide methods useful in the present disclosure are wellknown to those skilled in the art.

For example, mutagenesis and directed evolution methods can be readilyapplied to polynucleotides to generate variant libraries that can beexpressed, screened, and assayed. Mutagenesis and directed evolutionmethods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548,6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201,6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344,6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160,6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742,6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246,6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855,6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224,6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011,6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603,6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098,6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514,6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922,6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515,7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347,7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170,7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428,7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499,7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674,8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681,8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871,9,593,326, and all related non-US counterparts; Ling et al., Anal.Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74[1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al.,Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986];Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323[1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999];Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al.,Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319[1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad.Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of whichare incorporated herein by reference).

In some embodiments, the polynucleotide encodes a transaminasepolypeptide comprising an amino acid sequence that is at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more identical to a reference sequence selected from theeven-numbered sequence identifiers of SEQ ID NO: 2, 4, 8, 366, and/or650, where the polypeptide has transaminase activity and one or more ofthe improved properties as described herein, for example the ability toconvert compound (1) to product compound (2) with increased activitycompared to a reference sequence (e.g., the polypeptide of SEQ ID NO: 2,4, 8, 366, and/or 650). In some embodiments, the reference sequence isselected from SEQ ID NO: 2, 4, 8, 366, and/or 650. In some embodiments,the reference sequence is SEQ ID NO: 2. In some embodiments, thereference sequence is SEQ ID NO: 4. In some embodiments, the referencesequence is SEQ ID NO: 8. In some embodiments, the reference sequence isSEQ ID NO: 366. In some embodiments, the reference sequence is SEQ IDNO: 650.

In some embodiments, the polynucleotide encodes an engineeredtransaminase polypeptide comprising an amino acid sequence that has thepercent identity described above and (a) has one or more amino acidresidue differences as compared to SEQ ID NO: 2, 4, 8, 366, and/or 650.In some embodiments, the present disclosure provides an engineeredpolypeptide having transaminase activity comprising an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity toreference sequence of SEQ ID NO: 2, 4, 8, 366, and/or 650 and (a) atleast one amino acid residue difference selected from thosesubstitutions provided herein (See e.g., Tables 2-1, 2-2, 3-1, 3-2, 4-1,4-2, 5-1, and/or 5-2).

In some embodiments, the polynucleotide encoding the engineeredtransaminase polypeptide comprises a sequence selected from theodd-numbered sequence identifiers of SEQ ID NO: 5-935. In someembodiments, the polynucleotide sequences are selected from SEQ ID NO:1, 3, 7, 365 and/or 935. In some embodiments, the present disclosureprovides engineered polynucleotides encoding polypeptides havingtransaminase activity, wherein the engineered polypeptides have at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to at least one reference sequenceselected from SEQ ID NO: 1, 3, 7, 365 and/or 935.

In some embodiments, the present disclosure provides a polynucleotidethat hybridizes under defined conditions, such as moderately stringentor highly stringent conditions, to a polynucleotide sequence (orcomplement thereof) encoding an engineered transaminase of the presentdisclosure. In some embodiments, the polynucleotides are capable ofhybridizing under highly stringent conditions to a polynucleotideselected from the sequences having the odd-numbered sequence identifiersof SEQ ID NO: 5-935, or a complement thereof, and encodes a polypeptidehaving transaminase activity with one or more of the improved propertiesdescribed herein.

In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes an engineered transaminasepolypeptide comprising an amino acid sequence that has one or more aminoacid residue differences as compared to SEQ ID NO:4 selected from 18,20, 21, 21/23/56/146, 21/23/56/146/432, 21/23/146/417,21/23/395/417/432, 21/53/56, 21/53/417, 21/56/395, 21/417/432, 23,23/53, 23/53/56, 23/53/56/146/395, 23/53/395, 23/53/417, 23/53/432,23/56, 23/56/395, 23/56/395/417, 23/395/417, 23/417, 23/417/432, 53,53/56, 53/146/417, 53/395, 56, 56/74/241/286/314/316/323,56/86/163/314/316/383/414/416/422, 56/86/286/314/414/416,56/86/314/316/323/394/414/422, 56/146/417, 56/146/432, 56/147, 56/163,56/163/286/316/323/383/394, 56/286/314/316/323/422, 56/286/383, 56/323,56/323/383, 56/323/383/394, 56/383, 56/395, 74/81/286/316/323/383,74/85/86/163/286/316/323/394, 74/85/314/316/414/416, 74/86/163/316,74/86/316/323/383/394, 74/88/286/316/323/383, 74/88/323/383,74/163/286/316/383/394/416, 74/163/314/316, 74/163/314/316/323/394,74/163/314/323/383/414/416, 74/286, 74/286/316/323, 74/286/394/416,74/314/323/383/394/414, 74/316/323/394, 85/86/88/163/323/383/394,85/86/163/314/323/394/414, 85/286, 85/286/323, 86,86/88/163/323/383/414/422, 86/383/394, 88, 88/163/286/383,88/286/316/323, 88/286/316/323/383/414/416, 88/316/323, 146,146/147/395/417, 146/395, 146/395/417, 146/417, 147/395/417/432,147/417, 149, 157, 163, 163/222/286/316/323/383/394, 163/286,163/286/314/316/323/414/416, 163/286/314/323/394,163/286/316/323/394/416, 163/286/414, 163/314/316/394, 163/314/323/394,163/314/383, 163/314/414, 163/316/323, 163/323, 163/383, 164, 199/417,259, 260, 284, 286, 286/314/323/383, 286/314/394,286/316/323/383/414/416, 286/316/383/394, 286/316/394/414/416, 286/323,286/323/383/414, 286/323/416, 286/383, 286/416, 314/316, 314/316/323,314/316/323/383/422, 314/316/323/394, 314/316/394, 314/323/383/394,314/383, 314/383/414/422, 315, 316, 316/323/383/394,316/323/394/414/416, 316/414/422, 323, 323/383, 323/383/394/414/416,323/394, 383, 395, 395/417, 395/417/432, 400, 401, 403, 404, 405, 406,408, 415, 417, 417/432, 420, and 422, wherein the positions are numberedwith reference to SEQ ID NO:4. In some embodiments, the amino aciddifferences comprise the substitution(s) 18A, 20C, 21H,21P/23S/56C/146H, 21P/23S/56C/146H/432V, 21P/23S/146H/417V,21P/23S/395D/417S/432V, 21P/53C/56C, 21P/53C/417S, 21P/56C/395D,21P/417S/432V, 21R, 23A, 23R, 23S/53C, 23S/53C/56C,23S/53C/56C/146H/395D, 23S/53C/395D, 23S/53C/417S, 23S/53C/432V,23S/56C, 23S/56C/395D, 23S/56C/395D/417V, 23S/395D/417S, 23S/417S,23S/417V, 23S/417V/432V, 53C, 53C/56C, 53C/146H/417S, 53C/395D, 56A,56A/74T/241V/286S/314R/316W/323T,56A/86A/163F/314R/316W/383V/414V/416A/422A, 56A/86A/286S/314R/414V/416A,56A/86A/314R/316W/323T/394G/414V/422A, 56A/163F,56A/163F/286S/316W/323T/383V/394G, 56A/286S/314R/316W/323T/422A,56A/286S/383V, 56A/323T, 56A/323T/383V, 56A/323T/383V/394G, 56A/383V,56C, 56C/146H/417V, 56C/146H/432V, 56C/147R, 56C/395D, 56T, 56V,74T/81S/286S/316W/323T/383V, 74T/85V/86A/163F/286S/316W/323T/394G,74T/85V/314R/316W/414V/416A, 74T/86A/163F/316W,74T/86A/316W/323T/383V/394G, 74T/88R/286S/316W/323T/383V,74T/88R/323T/383V, 74T/163F/286S/316W/383V/394G/416A,74T/163F/314R/316W, 74T/163F/314R/316W/323T/394G,74T/163F/314R/323T/383V/414V/416A, 74T/286S, 74T/286S/316W/323T,74T/286S/394G/416A, 74T/314R/323T/383V/394G/414V, 74T/316W/323T/394G,85V/86A/88R/163F/323T/383V/394G, 85V/86A/163F/314R/323T/394G/414V,85V/286S, 85V/286S/323T, 86A/88R/163F/323T/383V/414V/422A,86A/383V/394G, 86G, 88R/163F/286S/383V, 88R/286S/316W/323T,88R/286S/316W/323T/383V/414V/416A, 88R/316W/323T, 88S, 88T, 146H,146H/147R/395D/417S, 146H/395D, 146H/395D/417S, 146H/417S, 146H/417V,147R/395D/417S/432V, 147R/417S, 149S, 157A, 163F,163F/222V/286S/316W/323T/383V/394G, 163F/286S,163F/286S/314R/316W/323T/414V/416A, 163F/286S/314R/323T/394G,163F/286S/316W/323T/394G/416A, 163F/286S/414V, 163F/314R/316W/394G,163F/314R/323T/394G, 163F/314R/383V, 163F/314R/414V, 163F/316W/323T,163F/323T, 163F/383V, 163L, 163M, 164A, 164D, 164Q, 164S, 199V/417S,259V, 260T, 284A, 286S, 286S/314R/323T/383V, 286S/314R/394G,286S/316W/323T/383V/414V/416A, 286S/316W/383V/394G,286S/316W/394G/414V/416A, 286S/323T, 286S/323T/383V/414V,286S/323T/416A, 286S/383V, 286S/416A, 314R/316W, 314R/316W/323T,314R/316W/323T/383V/422A, 314R/316W/323T/394G, 314R/316W/394G,314R/323T/383V/394G, 314R/383V, 314R/383V/414V/422A, 315G, 315R, 316A,316F, 316G, 316H, 316L, 316N, 316R, 316V, 316W/323T/383V/394G,316W/323T/394G/414V/416A, 316W/414V/422A, 323C, 323S, 323T, 323T/383V,323T/383V/394G/414V/416A, 323T/394G, 383V, 395D, 395D/417S,395D/417S/432V, 395D/417V, 400D, 401A, 401K, 401S, 403V, 404S, 405H,405W, 406S, 408F, 408L, 408W, 415G, 415W, 417A, 417S, 417S/432V, 417V,417V/432V, 420G, 422L, and 422T, wherein the positions are numbered withreference to SEQ ID NO:4. In some additional embodiments, the amino aciddifferences comprise the substitution(s) G18A, T20C, D21H,D21P/P23S/L56C/R146H, D21P/P23S/L56C/R146H/A432V, D21P/P23S/R146H/L417V,D21P/P23S/G395D/L417S/A432V, D21P/N53C/L56C, D21P/N53C/L417S,D21P/L56C/G395D, D21P/L417S/A432V, D21R, P23A, P23R, P23S/N53C,P23S/N53C/L56C, P23S/N53C/L56C/R146H/G395D, P23S/N53C/G395D,P23S/N53C/L417S, P23S/N53C/A432V, P23S/L56C, P23S/L56C/G395D,P23S/L56C/G395D/L417V, P23S/G395D/L417S, P23S/L417S, P23S/L417V,P23S/L417V/A432V, N53C, N53C/L56C, N53C/R146H/L417S, N53C/G395D, L56A,L56A/A74T/A241V/N286S/I314R/E316W/A323T,L56A/S86A/K163F/I314R/E316W/A383V/C414V/P416A/V422A,L56A/S86A/N286S/I314R/C414V/P416A,L56A/S86A/I314R/E316W/A323T/D394G/C414V/V422A, L56A/K163F,L56A/K163F/N286S/E316W/A323T/A383V/D394G,L56A/N286S/I314R/E316W/A323T/V422A, L56A/N286S/A383V, L56A/A323T,L56A/A323T/A383V, L56A/A323T/A383V/D394G, L56A/A383V, L56C,L56C/R146H/L417V, L56C/R146H/A432V, L56C/W147R, L56C/G395D, L56T, L56V,A74T/G81S/N286S/E316W/A323T/A383V,A74T/F85V/S86A/K163F/N286S/E316W/A323T/D394G,A74T/F85V/I314R/E316W/C414V/P416A, A74T/S86A/K163F/E316W,A74T/S86A/E316W/A323T/A383V/D394G, A74T/H88R/N286S/E316W/A323T/A383V,A74T/H88R/A323T/A383V, A74T/K163F/N286S/E316W/A383V/D394G/P416A,A74T/K163F/I314R/E316W, A74T/K163F/I314R/E316W/A323T/D394G,A74T/K163F/I314R/A323T/A383V/C414V/P416A, A74T/N286S,A74T/N286S/E316W/A323T, A74T/N286S/D394G/P416A,A74T/I314R/A323T/A383V/D394G/C414V, A74T/E316W/A323T/D394G,F85V/S86A/H88R/K163F/A323T/A383V/D394G,F85V/S86A/K163F/I314R/A323T/D394G/C414V, F85V/N286S, F85V/N286S/A323T,S86A/H88R/K163F/A323T/A383V/C414V/V422A, S86A/A383V/D394G, S86G,H88R/K163F/N286S/A383V, H88R/N286S/E316W/A323T,H88R/N286S/E316W/A323T/A383V/C414V/P416A, H88R/E316W/A323T, H88S, H88T,R146H, R146H/W147R/G395D/L417S, R146H/G395D, R146H/G395D/L417S,R146H/L417S, R146H/L417V, W147R/G395D/L417S/A432V, W147R/L417S, A149S,S157A, K163F, K163F/A222V/N286S/E316W/A323T/A383V/D394G, K163F/N286S,K163F/N286S/I314R/E316W/A323T/C414V/P416A,K163F/N286S/I314R/A323T/D394G, K163F/N286S/E316W/A323T/D394G/P416A,K163F/N286S/C414V, K163F/I314R/E316W/D394G, K163F/I314R/A323T/D394G,K163F/I314R/A383V, K163F/I314R/C414V, K163F/E316W/A323T, K163F/A323T,K163F/A383V, K163L, K163M, P164A, P164D, P164Q, P164S, A199V/L417S,I259V, C260T, S284A, N286S, N286S/I314R/A323T/A383V, N286S/I314R/D394G,N286S/E316W/A323T/A383V/C414V/P416A, N286S/E316W/A383V/D394G,N286S/E316W/D394G/C414V/P416A, N286S/A323T, N286S/A323T/A383V/C414V,N286S/A323T/P416A, N286S/A383V, N286S/P416A, I314R/E316W,I314R/E316W/A323T, I314R/E316W/A323T/A383V/V422A,I314R/E316W/A323T/D394G, I314R/E316W/D394G, I314R/A323T/A383V/D394G,I314R/A383V, I314R/A383V/C414V/V422A, E315G, E315R, E316A, E316F, E316G,E316H, E316L, E316N, E316R, E316V, E316W/A323T/A383V/D394G,E316W/A323T/D394G/C414V/P416A, E316W/C414V/V422A, A323C, A323S, A323T,A323T/A383V, A323T/A383V/D394G/C414V/P416A, A323T/D394G, A383V, G395D,G395D/L417S, G395D/L417S/A432V, G395D/L417V, S400D, E401A, E401K, E401S,I403V, A404S, N405H, N405W, T406S, T408F, T408L, T408W, R415G, R415W,L417A, L417S, L417S/A432V, L417V, L417V/A432V, S420G, V422L, and V422T,wherein the positions are numbered with reference to SEQ ID NO: 4.

In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes an engineered transaminasepolypeptide comprising an amino acid sequence that has one or more aminoacid residue differences as compared to SEQ ID NO: 4 selected from74/81/286/316/323/383, 163/286/314/316/323/414/416, 163/286/314/323/394,286/314/323/383, 286/316/323/383/414/416, 315, and 408, wherein thepositions are numbered with reference to SEQ ID NO: 4. In someembodiments, the amino acid differences comprise the substitution(s)74T/81S/286S/316W/323T/383V, 163F/286S/314R/316W/323T/414V/416A,163F/286S/314R/323T/394G, 286S/314R/323T/383V,286S/316W/323T/383V/414V/416A, 315G, and 408F, wherein the positions arenumbered with reference to SEQ ID NO: 4. In some additional embodiments,the amino acid differences comprise the substitution(s)A74T/G81S/N286S/E316W/A323T/A383V,K163F/N286S/I314R/E316W/A323T/C414V/P416A,K163F/N286S/I314R/A323T/D394G, N286S/I314R/A323T/A383V,N286S/E316W/A323T/A383V/C414V/P416A, E315G, and T408F, wherein thepositions are numbered with reference to SEQ ID NO: 4.

In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes an engineered transaminasepolypeptide comprising an amino acid sequence that has one or more aminoacid residue differences as compared to SEQ ID NO: 8 selected from 5,18/23/149/260/383/395/401/416, 18/23/149/383, 18/163/164, 21,21/163/315/316, 21/163/323/408, 21/408,23/56/86/149/163/164/383/401/416, 23/86, 23/149/260, 23/149/284/383/395,23/163/164/383, 23/163/164/401/416, 24, 42, 42/110, 42/187/272,42/187/324/363/366, 42/187/353, 42/272/291, 42/272/291/363,42/272/324/363/366, 42/272/363/410, 42/272/410, 42/291/313/363/410,42/291/363, 42/291/363/366, 42/353, 42/363, 46, 66, 77,86/149/163/164/383/395/401, 86/149/395, 86/163/164/260/383, 86/383, 107,110, 110/187, 110/187/253/410, 134, 138, 149/164/260/383/395/401,149/260/383, 149/416, 163/259/323/408, 163/259/408, 163/315/316,164/260/401, 164/316/383/401, 167, 186, 187, 187/253/363/366,187/272/324/363/410, 187/272/363, 187/272/363/366/410, 187/291, 189,191, 195, 199, 203, 210, 211, 248, 259/307, 260/395/401, 272, 272/353,272/363/366, 272/410, 277, 291, 305, 309, 315, 342, 343, 351, 354, 358,361, 362, 363, 363/366, 365, 367, 383, 383/401, 383/416/422, 385, 388,389, 392, 395, 396, 401, 404, 405, 408, 410, 416, 417, 439, 443, 447,450, and 451, wherein the positions are numbered with reference to SEQID NO: 8. In some embodiments, the amino acid differences comprise thesubstitution(s) 5E, 5G, 18A/23R/149S/260T/383V/395D/401S/416P,18A/23R/149S/383V, 18A/163M/164Q, 21H, 21H/163L/315G/316F,21H/163L/323C/408F, 21H/408F, 23R/56C/86G/149S/163M/164D/383V/401S/416P,23R/86G, 23R/149S/260T, 23R/149S/284A/383V/395D, 23R/163M/164Q/383V,23R/163M/164S/401S/416P, 24K, 24R, 42F, 42F/110K, 42F/187E/272E,42F/187E/324S/363L/366H, 42F/187E/353T, 42F/272E/291Y,42F/272E/291Y/363L, 42F/272E/324S/363L/366H, 42F/272E/363L/410H,42F/272E/410H, 42F/291Y/313V/363L/410H, 42F/291Y/363L,42F/291Y/363L/366H, 42F/353T, 42F/363L, 46S, 66A, 77M,86G/149S/163M/164S/383V/395D/401S, 86G/149S/395D,86G/163M/164S/260T/383V, 86G/383V, 107L, 107S, 107Y, 110K, 110K/187E,110K/187E/253L/410H, 134V, 138R, 149S/164S/260T/383V/395D/401A,149S/260T/383V, 149S/416P, 163L/259V/323C/408F, 163L/259V/408F,163L/315G/316F, 164D/316H/383V/401S, 164S/260T/401S, 167N, 186Q, 187E,187E/253L/363L/366H, 187E/272E/324S/363L/410H, 187E/272E/363L,187E/272E/363L/366H/410H, 187E/291Y, 189F, 189S, 189V, 189W, 191D, 191F,195W, 199Q, 203L, 210A, 210L, 210M, 210V, 210Y, 211R, 248G, 259V/307M,260T/395D/401S, 272E, 272E/353T, 272E/363L/366H, 272E/410H, 277S, 291Y,305E, 309A, 309F, 309R, 315G, 342T, 343G, 351L, 354S, 358L, 361R, 362Q,362V, 363L, 363L/366H, 365L, 365Q, 365R, 365S, 367T, 383V, 383V/401A,383V/416P/422T, 385L, 385T, 388D, 388L, 388P, 389D, 392A, 392L, 395R,396P, 396Y, 401Q, 401S, 404M, 405W, 408A, 408E, 408W, 410H, 416P, 417S,439L, 439S, 443L, 443S, 447S, 447T, 450D, and 451S, wherein thepositions are numbered with reference to SEQ ID NO: 8. In someadditional embodiments, the amino acid differences comprise thesubstitution(s) QSE, QSG, G18A/P23R/A149S/C260T/A383V/G395D/E401S/A416P,G18A/P23R/A149S/A383V, G18A/F163M/P164Q, D21H, D21H/F163L/E315G/W316F,D21H/F163L/T323C/T408F, D21H/T408F,P23R/L56C/S86G/A149S/F163M/P164D/A383V/E401S/A416P, P23R/S86G,P23R/A149S/C260T, P23R/A149S/S284A/A383V/G395D, P23R/F163M/P164Q/A383V,P23R/F163M/P164S/E401S/A416P, S24K, S24R, V42F, V42F/R110K,V42F/Y187E/V272E, V42F/Y187E/G324S/I363L/R366H, V42F/Y187E/A353T,V42F/V272E/F291Y, V42F/V272E/F291Y/I363L, V42F/V272E/G324S/I363L/R366H,V42F/V272E/I363L/L410H, V42F/V272E/L410H, V42F/F291Y/A313V/I363L/L410H,V42F/F291Y/I363L, V42F/F291Y/I363L/R366H, V42F/A353T, V42F/I363L, G46S,K66A, E77M, S86G/A149S/F163M/P164S/A383V/G395D/E401S, S86G/A149S/G395D,S86G/F163M/P164S/C260T/A383V, S86G/A383V, D107L, D107S, D107Y, R110K,R110K/Y187E, R110K/Y187E/V253L/L410H, A134V, P138R,A149S/P164S/C260T/A383V/G395D/E401A, A149S/C260T/A383V, A149S/A416P,F163L/I259V/T323C/T408F, F163L/I259V/T408F, F163L/E315G/W316F,P164D/W316H/A383V/E401S, P164S/C260T/E401S, S167N, R186Q, Y187E,Y187E/V253L/I363L/R366H, Y187E/V272E/G324S/I363L/L410H,Y187E/V272E/I363L, Y187E/V272E/I363L/R366H/L410H, Y187E/F291Y, E189F,E189S, E189V, E189W, G191D, G191F, E195W, A199Q, R203L, Q210A, Q210L,Q210M, Q210V, Q210Y, K211R, K248G, I259V/L307M, C260T/G395D/E401S,V272E, V272E/A353T, V272E/I363L/R366H, V272E/L410H, T277S, F291Y, K305E,T309A, T309F, T309R, E315G, N342T, E343G, R351L, P354S, E358L, K361R,H362Q, H362V, I363L, I363L/R366H, E365L, E365Q, E365R, E365S, P367T,A383V, A383V/E401A, A383V/A416P/V422T, K385L, K385T, A388D, A388L,A388P, S389D, P392A, P392L, G395R, N396P, N396Y, E401Q, E401S, A404M,N405W, T408A, T408E, T408W, L410H, A416P, L417S, D439L, D439S, K443L,K443S, K447S, K447T, A450D, and E451S, wherein the positions arenumbered with reference to SEQ ID NO: 8.

In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes an engineered transaminasepolypeptide comprising an amino acid sequence that has one or more aminoacid residue differences as compared to SEQ ID NO: 8 selected from18/23/149/383, 21/163/323/408, 272, 291, and 383, wherein the positionsare numbered with reference to SEQ ID NO: 8. In some embodiments, theamino acid differences comprise the substitution(s) 18A/23R/149S/383V,21H/163L/323C/408F, 272E, 291Y, and 383V, wherein the positions arenumbered with reference to SEQ ID NO: 8. In some additional embodiments,the amino acid differences comprise the substitution(s)G18A/P23R/A149S/A383V, D21H/F163L/T323C/T408F, V272E, F291Y, and A383V,wherein the positions are numbered with reference to SEQ ID NO: 8.

In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes an engineered transaminasepolypeptide comprising an amino acid sequence that has one or more aminoacid residue differences as compared to SEQ ID NO: 366 selected from 24,24/42/66/291, 24/42/291/362, 24/66/163/191/362/383/388,24/66/191/199/260/291/351, 24/66/191/199/291, 24/66/191/260/408,24/66/260/291/383/388/408, 24/66/291/342/383, 24/66/291/365,24/66/342/365/388/408, 24/77/291, 24/107/163/191/291/351/383/388,24/107/291/351/365/388, 24/163/351/383, 24/191/291/365,24/199/260/351/362/383, 24/199/260/362/383/388, 24/260/362/383/388,24/291, 24/291/342/351/383, 24/291/362/388, 24/291/408, 24/383/388,24/388, 25, 28, 33, 42/191/408, 42/199/291/383,42/291/351/362/365/383/388, 42/291/351/362/383/408, 42/291/383/388,66/82/291/383, 66/163/191/365/383, 66/199/351/383, 66/291,66/291/362/365/383, 66/291/383/388, 66/383, 77/291, 77/383/388, 86,107/191/199/365/383/388, 107/191/291/383, 148, 153,163/291/362/365/383/388, 163/291/383/388, 163/383, 191/199/365/383/388,191/260/388, 191/291, 191/291/342/362/365, 191/351/383/388, 199/260/383,199/291, 260, 260/291/365/383/408, 260/365/383, 291, 291/351/383/388,291/351/383/388/408, 291/362/365, 291/365/388, 291/383, 314, 315, 316,319, 342/362, 351/383/388, 362, 362/388, 383, 383/388, 396, 397, 405,406, 413, 419, and 423, wherein the positions are numbered withreference to SEQ ID NO: 366. In some embodiments, the amino aciddifferences comprise the substitution(s) 24E, 24K, 24K/42F/66A/291Y,24K/42F/291Y/362Q, 24K/66A/163M/191D/362Q/383V/388D,24K/66A/191D/199Q/260T/291Y/351L, 24K/66A/191D/199Q/291Y,24K/66A/191D/260T/408A, 24K/66A/260T/291Y/383V/388D/408A,24K/66A/291Y/342T/383V, 24K/66A/291Y/365S, 24K/66A/342T/365S/388D/408E,24K/77M/291Y, 24K/107L/163M/191D/291Y/351L/383V/388D,24K/107L/291Y/351L/365S/388D, 24K/163M/351L/383V, 24K/191D/291Y/365S,24K/199Q/260T/351L/362Q/383V, 24K/199Q/260T/362Q/383V/388D,24K/260T/362Q/383V/388D, 24K/291Y, 24K/291Y/342T/351L/383V,24K/291Y/362Q/388D, 24K/291Y/408A, 24K/383V/388D, 24K/388D, 25H, 25V,28S, 28T, 33T, 42F/191D/408E, 42F/199Q/291Y/383V,42F/291Y/351L/362Q/365S/383V/388D, 42F/291Y/351L/362Q/383V/408A,42F/291Y/383V/388D, 66A/82H/291Y/383V, 66A/163M/191D/365S/383V,66A/199Q/351L/383V, 66A/291Y, 66A/291Y/362Q/365S/383V,66A/291Y/383V/388D, 66A/383V, 77M/291Y, 77M/383V/388D, 86T,107L/191D/199Q/365S/383V/388D, 107L/191D/291Y/383V, 148G, 153S,163M/291Y/362Q/365S/383V/388D, 163M/291Y/383V/388D, 163M/383V,191D/199Q/365S/383V/388D, 191D/260T/388D, 191D/291Y,191D/291Y/342T/362Q/365S, 191D/351L/383V/388D, 199Q/260T/383V,199Q/291Y, 260T, 260T/291Y/365S/383V/408A, 260T/365S/383V, 291Y,291Y/351L/383V/388D, 291Y/351L/383V/388D/408A, 291Y/362Q/365S,291Y/365S/388D, 291Y/383V, 314K, 315S, 316V, 319S, 342T/362Q,351L/383V/388D, 362Q, 362Q/388D, 383V, 383V/388D, 396R, 397M, 405A,406H, 413L, 419S, and 423V, wherein the positions are numbered withreference to SEQ ID NO: 366. In some additional embodiments, the aminoacid differences comprise the substitution(s) S24E, S24K,S24K/V42F/K66A/F291Y, S24K/V42F/F291Y/H362Q,S24K/K66A/L163M/G191D/H362Q/A383V/A388D,S24K/K66A/G191D/A199Q/C260T/F291Y/R351L, S24K/K66A/G191D/A199Q/F291Y,S24K/K66A/G191D/C260T/F408A, S24K/K66A/C260T/F291Y/A383V/A388D/F408A,S24K/K66A/F291Y/N342T/A383V, S24K/K66A/F291Y/E365S,S24K/K66A/N342T/E365S/A388D/F408E, S24K/E77M/F291Y,S24K/D107L/L163M/G191D/F291Y/R351L/A383V/A388D,S24K/D107L/F291Y/R351L/E365S/A388D, S24K/L163M/R351L/A383V,S24K/G191D/F291Y/E365S, S24K/A199Q/C260T/R351L/H362Q/A383V,S24K/A199Q/C260T/H362Q/A383V/A388D, S24K/C260T/H362Q/A383V/A388D,S24K/F291Y, S24K/F291Y/N342T/R351L/A383V, S24K/F291Y/H362Q/A388D,S24K/F291Y/F408A, S24K/A383V/A388D, S24K/A388D, L25H, L25V, R28S, R28T,V33T, V42F/G191D/F408E, V42F/A199Q/F291Y/A383V,V42F/F291Y/R351L/H362Q/E365S/A383V/A388D,V42F/F291Y/R351L/H362Q/A383V/F408A, V42F/F291Y/A383V/A388D,K66A/Y82H/F291Y/A383V, K66A/L163M/G191D/E365S/A383V,K66A/A199Q/R351L/A383V, K66A/F291Y, K66A/F291Y/H362Q/E365S/A383V,K66A/F291Y/A383V/A388D, K66A/A383V, E77M/F291Y, E77M/A383V/A388D, S86T,D107L/G191D/A199Q/E365S/A383V/A388D, D107L/G191D/F291Y/A383V, N148G,A153S, L163M/F291Y/H362Q/E365S/A383V/A388D, L163M/F291Y/A383V/A388D,L163M/A383V, G191D/A199Q/E365S/A383V/A388D, G191D/C260T/A388D,G191D/F291Y, G191D/F291Y/N342T/H362Q/E365S, G191D/R351L/A383V/A388D,A199Q/C260T/A383V, A199Q/F291Y, C260T, C260T/F291Y/E365S/A383V/F408A,C260T/E365S/A383V, F291Y, F291Y/R351L/A383V/A388D,F291Y/R351L/A383V/A388D/F408A, F291Y/H362Q/E365S, F291Y/E365S/A388D,F291Y/A383V, R314K, E315S, W316V, H319S, N342T/H362Q, R351L/A383V/A388D,H362Q, H362Q/A388D, A383V, A383V/A388D, N396R, L397M, N405A, T406H,I413L, Q419S, and L423V, wherein the positions are numbered withreference to SEQ ID NO: 366.

In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes an engineered transaminasepolypeptide comprising an amino acid sequence that has one or more aminoacid residue differences as compared to SEQ ID NO: 366 selected from24/66/191/199/291, 24/66/291/365, 163/291/362/365/383/388,163/291/383/388, 191/291/342/362/365, 291, and 291/383, wherein thepositions are numbered with reference to SEQ ID NO: 366. In someembodiments, the amino acid differences comprise the substitution(s)24K/66A/191D/199Q/291Y, 24K/66A/291Y/365S,163M/291Y/362Q/365S/383V/388D, 163M/291Y/383V/388D,191D/291Y/342T/362Q/365S, 291Y, and 291Y/383V, wherein the positions arenumbered with reference to SEQ ID NO: 366. In some additionalembodiments, the amino acid differences comprise the substitution(s)S24K/K66A/G191D/A199Q/F291Y, S24K/K66A/F291Y/E365S,L163M/F291Y/H362Q/E365S/A383V/A388D, L163M/F291Y/A383V/A388D,G191D/F291Y/N342T/H362Q/E365S, F291Y, and F291Y/A383V, wherein thepositions are numbered with reference to SEQ ID NO: 366.

In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes an engineered transaminasepolypeptide comprising an amino acid sequence that has one or more aminoacid residue differences as compared to SEQ ID NO: 650 selected from 10,13, 13/24/108/163, 13/24/108/163/311, 13/24/133/199/311, 13/24/163,13/24/199/311, 13/108, 13/108/199, 13/108/311, 13/199, 13/311, 14,14/24/108, 14/24/108/133, 14/24/108/199, 14/24/199, 14/108,14/108/133/311, 14/108/311, 14/311, 24, 24/163, 24/163/199, 35, 72, 73,78, 95, 101, 108, 108/199, 114, 154, 163, 169, 175/316, 199, 199/311,226, 293, 311, 316, 382, 383, and 386, wherein the positions arenumbered with reference to SEQ ID NO: 650. In some embodiments, theamino acid differences comprise the substitution(s) 10E, 13A,13A/24E/108R/163L/311S, 13A/24E/133R/199Q/311S, 13A/24E/163L,13A/24K/108R/163L, 13A/24K/199Q/311S, 13A/108R, 13A/108R/199Q,13A/108R/311S, 13A/199Q, 13A/311S, 14A, 14G, 14H, 14H/24E/108R/133R,14H/24K/108R, 14H/24K/108R/199Q, 14H/24K/199Q, 14H/108R,14H/108R/133R/311S, 14H/108R/311S, 14H/311S, 24E, 24E/163L,24E/163L/199Q, 35E, 72G, 73R, 73S, 78A, 95I, 101L, 108R, 108R/199Q,114A, 154S, 163H, 163S, 163V, 169C, 169V, 175D/316F, 199Q, 199Q/311S,226Q, 293A, 311K, 316D, 316E, 316F, 316G, 316H, 316I, 316L, 316N, 316S,316V, 316Y, 382D, 383L, and 386A, wherein the positions are numberedwith reference to SEQ ID NO: 650. In some additional embodiments, theamino acid differences comprise the substitution(s) R10E, T13A,T13A/S24E/S108R/M163L/I311S, T13A/S24E/A133R/A199Q/I311S,T13A/S24E/M163L, T13A/S24K/S108R/M163L, T13A/S24K/A199Q/I311S,T13A/S108R, T13A/S108R/A199Q, T13A/S108R/I311S, T13A/A199Q, T13A/I311S,Y14A, Y14G, Y14H, Y14H/S24E/S108R/A133R, Y14H/S24K/S108R,Y14H/S24K/S108R/A199Q, Y14H/S24K/A199Q, Y14H/S108R,Y14H/S108R/A133R/I311S, Y14H/S108R/I311S, Y14H/I311S, S24E, S24E/M163L,S24E/M163L/A199Q, H35E, A72G, K73R, K73S, R78A, M95I, V101L, S108R,S108R/A199Q, T114A, T154S, M163H, M163S, M163V, F169C, F169V,G175D/W316F, A199Q, A199Q/1311S, M226Q, P293A, I311K, W316D, W316E,W316F, W316G, W316H, W316I, W316L, W316N, W316S, W316V, W316Y, E382D,V383L, and D386A, wherein the positions are numbered with reference toSEQ ID NO: 650.

In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes an engineered transaminasepolypeptide comprising an amino acid sequence that has one or more aminoacid residue differences as compared to SEQ ID NO: 650 selected from14/108/133/311, 24/163/199, 72, 78, 316, and 383, wherein the positionsare numbered with reference to SEQ ID NO: 650. In some embodiments, theamino acid differences comprise the substitution(s) 14H/108R/133R/311S,24E/163L/199Q, 72G, 78A, 316L, 316N, 316S, and 383L, wherein thepositions are numbered with reference to SEQ ID NO: 650. In someadditional embodiments, the amino acid differences comprise thesubstitution(s) Y14H/S108R/A133R/I311S, S24E/M163L/A199Q, A72G, R78A,W316L, W316N, W316S, and V383L, wherein the positions are numbered withreference to SEQ ID NO: 650.

In some embodiments, the variant transaminase of the present disclosurefurther comprises additional sequences that do not alter the encodedactivity of the enzyme. For example, in some embodiments, the varianttransaminase is linked to an epitope tag or to another sequence usefulin purification.

In some embodiments, the variant transaminase polypeptides of thepresent disclosure are secreted from the host cell in which they areexpressed (e.g., a yeast or filamentous fungal host cell) and areexpressed as a pre-protein including a signal peptide (i.e., an aminoacid sequence linked to the amino terminus of a polypeptide and whichdirects the encoded polypeptide into the cell secretory pathway).

When the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical ligationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of thedisclosure can be prepared by chemical synthesis (e.g., using theclassical phosphoramidite method described by Beaucage et al., Tet.Lett., 22:1859-69 [1981], or the method described by Matthes et al.,EMBO J., 3:801-05 [1984], as it is typically practiced in automatedsynthetic methods). According to the phosphoramidite method,oligonucleotides are synthesized (e.g., in an automatic DNAsynthesizer), purified, annealed, ligated and cloned in appropriatevectors. In addition, essentially any nucleic acid can be obtained fromany of a variety of commercial sources (e.g., The Midland CertifiedReagent Company, Midland, TX, The Great American Gene Company, Ramona,CA, ExpressGen Inc. Chicago, IL, Operon Technologies Inc., Alameda, CA,and many others).

The present disclosure also provides recombinant constructs comprising asequence encoding at least one variant transaminase, as provided herein.In some embodiments, the present disclosure provides an expressionvector comprising a variant transaminase polynucleotide operably linkedto a heterologous promoter. In some embodiments, expression vectors ofthe present disclosure are used to transform appropriate host cells topermit the host cells to express the variant transaminase protein.Methods for recombinant expression of proteins in fungi and otherorganisms are well known in the art, and a number of expression vectorsare available or can be constructed using routine methods. In someembodiments, nucleic acid constructs of the present disclosure comprisea vector, such as, a plasmid, a cosmid, a phage, a virus, a bacterialartificial chromosome (BAC), a yeast artificial chromosome (YAC), andthe like, into which a nucleic acid sequence of the disclosure has beeninserted. In some embodiments, polynucleotides of the present disclosureare incorporated into any one of a variety of expression vectorssuitable for expressing variant transaminase polypeptide(s). Suitablevectors include, but are not limited to chromosomal, nonchromosomal andsynthetic DNA sequences (e.g., derivatives of SV40), as well asbacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus,adeno-associated virus, retroviruses, and many others. Any suitablevector that transduces genetic material into a cell, and, if replicationis desired, which is replicable and viable in the relevant host findsuse in the present disclosure.

In some embodiments, the construct further comprises regulatorysequences, including but not limited to a promoter, operably linked tothe protein encoding sequence. Large numbers of suitable vectors andpromoters are known to those of skill in the art. Indeed, in someembodiments, in order to obtain high levels of expression in aparticular host it is often useful to express the variant transaminasesof the present disclosure under the control of a heterologous promoter.In some embodiments, a promoter sequence is operably linked to the 5′region of the variant transaminase coding sequence using any suitablemethod known in the art. Examples of useful promoters for expression ofvariant transaminases include, but are not limited to promoters fromfungi. In some embodiments, a promoter sequence that drives expressionof a gene other than a transaminase gene in a fungal strain finds use.As a non-limiting example, a fungal promoter from a gene encoding anendoglucanase may be used. In some embodiments, a promoter sequence thatdrives the expression of a transaminase gene in a fungal strain otherthan the fungal strain from which the transaminases were derived findsuse. Examples of other suitable promoters useful for directing thetranscription of the nucleotide constructs of the present disclosure ina filamentous fungal host cell include, but are not limited to promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease (See e.g., WO 96/00787, incorporatedherein by reference), as well as the NA2-tpi promoter (a hybrid of thepromoters from the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase), promoters such as cbh1,cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (See e.g.,Nunberg et al., Mol. Cell Biol., 4:2306-2315 [1984]; Boel et al., EMBOJ., 3:1581-85 [1984]; and European Patent Appln. 137280, all of whichare incorporated herein by reference), and mutant, truncated, and hybridpromoters thereof.

In yeast host cells, useful promoters include, but are not limited tothose from the genes for Saccharomyces cerevisiae enolase (eno-1),Saccharomyces cerevisiae galactokinase (gall), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and S. cerevisiae 3-phosphoglycerate kinase. Additionaluseful promoters useful for yeast host cells are known in the art (Seee.g., Romanos et al., Yeast 8:423-488 [1992], incorporated herein byreference). In addition, promoters associated with chitinase productionin fungi find use in the present disclosure (See e.g., Blaiseau andLafay, Gene 120243-248 [1992]; and Limon et al., Curr. Genet., 28:478-83[1995], both of which are incorporated herein by reference).

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present disclosure, include butare not limited to the promoters obtained from the E. coli lac operon,E. coli trp operon, bacteriophage lambda, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc.Natl. Acad. Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter(See e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 [1983]).

In some embodiments, cloned variant transaminases of the presentdisclosure also have a suitable transcription terminator sequence, asequence recognized by a host cell to terminate transcription. Theterminator sequence is operably linked to the 3′ terminus of the nucleicacid sequence encoding the polypeptide. Any terminator that isfunctional in the host cell of choice finds use in the presentdisclosure. Exemplary transcription terminators for filamentous fungalhost cells include, but are not limited to those obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease (Seee.g., U.S. Pat. No. 7,399,627, incorporated herein by reference). Insome embodiments, exemplary terminators for yeast host cells includethose obtained from the genes for Saccharomyces cerevisiae enolase,Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomycescerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other usefulterminators for yeast host cells are well-known to those skilled in theart (See e.g., Romanos et al., Yeast 8:423-88 [1992]).

In some embodiments, a suitable leader sequence is part of a clonedvariant transaminase sequence, which is a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′ terminus of the nucleic acidsequence encoding the polypeptide. Any leader sequence that isfunctional in the host cell of choice finds use in the presentdisclosure. Exemplary leaders for filamentous fungal host cells include,but are not limited to those obtained from the genes for Aspergillusoryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.Suitable leaders for yeast host cells include, but are not limited tothose obtained from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase,Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP).

In some embodiments, the sequences of the present disclosure alsocomprise a polyadenylation sequence, which is a sequence operably linkedto the 3′ terminus of the nucleic acid sequence and which, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice finds use in the presentdisclosure. Exemplary polyadenylation sequences for filamentous fungalhost cells include, but are not limited to those obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Fusarium oxysporumtrypsin-like protease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are known in the art (Seee.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990 [1995]).

In some embodiments, the control sequence comprises a signal peptidecoding region encoding an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion that is foreign to the coding sequence. The foreign signalpeptide coding region may be required where the coding sequence does notnaturally contain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present disclosure.

In some embodiments, the signal peptide is an endogenous V. fluvialistransaminase signal peptide. In some additional embodiments, signalpeptides from other V. fluvialis secreted proteins are used. In someembodiments, other signal peptides find use, depending on the host celland other factors.

Effective signal peptide coding regions for bacterial host cellsinclude, but are not limited to the signal peptide coding regionsobtained from the genes for Bacillus NCIB 11837 maltogenic amylase,Bacillus stearothermophilus alpha-amylase, Bacillus licheniformissubtilisin, Bacillus licheniformis beta-lactamase, Bacillusstearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillussubtilis prsA. Further signal peptides are known in the art (See e.g.,Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993]).

Effective signal peptide coding regions for filamentous fungal hostcells include, but are not limited to the signal peptide coding regionsobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheiaspartic proteinase, Humicola insolens cellulase, and Humicolalanuginosa lipase.

Useful signal peptides for yeast host cells include, but are not limitedto genes for Saccharomyces cerevisiae alpha-factor and Saccharomycescerevisiae invertase. Other useful signal peptide coding regions areknown in the art (See e.g., Romanos et al., [1992], supra).

In some embodiments, the control sequence comprises a propeptide codingregion that codes for an amino acid sequence positioned at the aminoterminus of a polypeptide. The resultant polypeptide is known as aproenzyme or propolypeptide (or a zymogen in some cases). Apropolypeptide is generally inactive and can be converted to a matureactive transaminase polypeptide by catalytic or autocatalytic cleavageof the propeptide from the propolypeptide. The propeptide coding regionmay be obtained from the genes for Bacillus subtilis alkaline protease(aprE), Bacillus subtilis neutral protease (nprT), Saccharomycescerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, andMyceliophthora thermophila lactase (See e.g., WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

In some embodiments, regulatory sequences are also used to allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude, but are not limited to the lac, tac, and trp operator systems.In yeast host cells, suitable regulatory systems include, as examples,the ADH2 system or GAL1 system. In filamentous fungi, suitableregulatory sequences include the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the transaminasepolypeptide of the present disclosure would be operably linked with theregulatory sequence.

Thus, in additional embodiments, the present disclosure providesrecombinant expression vectors comprising a polynucleotide encoding anengineered transaminase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. In some embodiments, the various nucleic acidand control sequences described above are joined together to produce arecombinant expression vector that may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, insome embodiments, the nucleic acid sequences are expressed by insertingthe nucleic acid sequence or a nucleic acid construct comprising thesequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector comprises any suitable vector (e.g., aplasmid or virus), that can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector typically depends on thecompatibility of the vector with the host cell into which the vector isto be introduced. In some embodiments, the vectors are linear or closedcircular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome). In some embodiments, thevector contains any means for assuring self-replication. Alternatively,in some other embodiments, upon being introduced into the host cell, thevector is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, inadditional embodiments, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon find use.

In some embodiments, the expression vector of the present disclosurecontains one or more selectable markers, which permit easy selection oftransformed cells. A “selectable marker” is a gene, the product of whichprovides for biocide or viral resistance, resistance to antimicrobialsor heavy metals, prototrophy to auxotrophs, and the like. Any suitableselectable markers for use in a filamentous fungal host cell find use inthe present disclosure, including, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Additional markers useful in host cells such as Aspergillus, include butare not limited to the amdS and pyrG genes of Aspergillus nidulans orAspergillus oryzae, and the bar gene of Streptomyces hygroscopicus.Suitable markers for yeast host cells include, but are not limited toADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Examples of bacterialselectable markers include, but are not limited to the dal genes fromBacillus subtilis or Bacillus licheniformis, or markers, which conferantibiotic resistance such as ampicillin, kanamycin, chloramphenicol,and or tetracycline resistance.

In some embodiments, the expression vectors of the present disclosurecontain an element(s) that permits integration of the vector into thehost cell's genome or autonomous replication of the vector in the cellindependent of the genome. In some embodiments involving integrationinto the host cell genome, the vectors rely on the nucleic acid sequenceencoding the polypeptide or any other element of the vector forintegration of the vector into the genome by homologous ornon-homologous recombination.

In some alternative embodiments, the expression vectors containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements preferably contain a sufficient number ofnucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000base pairs, and most preferably 800 to 10,000 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aon or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, or pAMβ1 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes its functioningtemperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl.Acad. Sci. USA 75:1433 [1978]).

In some embodiments, more than one copy of a nucleic acid sequence ofthe present disclosure is inserted into the host cell to increaseproduction of the gene product. An increase in the copy number of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by cultivating the cells in the presence of theappropriate selectable agent.

Many of the expression vectors for use in the present disclosure arecommercially available. Suitable commercial expression vectors include,but are not limited to the p3×FLAGTM™ expression vectors (Sigma-AldrichChemicals), which include a CMV promoter and hGH polyadenylation sitefor expression in mammalian host cells and a pBR322 origin ofreplication and ampicillin resistance markers for amplification in E.coli. Other suitable expression vectors include, but are not limited topBluescriptII SK(−) and pBK-CMV (Stratagene), and plasmids derived frompBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly(See e.g., Lathe et al., Gene 57:193-201 [1987]).

Thus, in some embodiments, a vector comprising a sequence encoding atleast one variant transaminase is transformed into a host cell in orderto allow propagation of the vector and expression of the varianttransaminase (s). In some embodiments, the variant transaminases arepost-translationally modified to remove the signal peptide and, in somecases, may be cleaved after secretion. In some embodiments, thetransformed host cell described above is cultured in a suitable nutrientmedium under conditions permitting the expression of the varianttransaminase(s). Any suitable medium useful for culturing the host cellsfinds use in the present disclosure, including, but not limited tominimal or complex media containing appropriate supplements. In someembodiments, host cells are grown in HTP media. Suitable media areavailable from various commercial suppliers or may be prepared accordingto published recipes (e.g., in catalogues of the American Type CultureCollection).

In another aspect, the present disclosure provides host cells comprisinga polynucleotide encoding an improved transaminase polypeptide providedherein, the polynucleotide being operatively linked to one or morecontrol sequences for expression of the transaminase enzyme in the hostcell. Host cells for use in expressing the transaminase polypeptidesencoded by the expression vectors of the present disclosure are wellknown in the art and include but are not limited to, bacterial cells,such as E. coli, Bacillus megaterium, Lactobacillus kefir, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells(e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; andplant cells. Appropriate culture media and growth conditions for theabove-described host cells are well known in the art.

Polynucleotides for expression of the transaminase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells areknown to those skilled in the art.

In some embodiments, the host cell is a eukaryotic cell. Suitableeukaryotic host cells include, but are not limited to, fungal cells,algal cells, insect cells, and plant cells. Suitable fungal host cellsinclude, but are not limited to, Ascomycota, Basidiomycota,Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, thefungal host cells are yeast cells and filamentous fungal cells. Thefilamentous fungal host cells of the present disclosure include allfilamentous forms of the subdivision Eumycotina and Oomycota.Filamentous fungi are characterized by a vegetative mycelium with a cellwall composed of chitin, cellulose and other complex polysaccharides.The filamentous fungal host cells of the present disclosure aremorphologically distinct from yeast.

In some embodiments of the present disclosure, the filamentous fungalhost cells are of any suitable genus and species, including, but notlimited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora,Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces,Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium,Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes,Tolypocladium, Trichoderma, Verticillium, and/or Volvariella, and/orteleomorphs, or anamorphs, and synonyms, basionyms, or taxonomicequivalents thereof.

In some embodiments of the present disclosure, the host cell is a yeastcell, including but not limited to cells of Candida, Hansenula,Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowiaspecies. In some embodiments of the present disclosure, the yeast cellis Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis,Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichiamethanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, orYarrowia lipolytica.

In some embodiments of the disclosure, the host cell is an algal cellsuch as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (e.g.,Phormidium sp. ATCC29409).

In some other embodiments, the host cell is a prokaryotic cell. Suitableprokaryotic cells include, but are not limited to Gram-positive,Gram-negative and Gram-variable bacterial cells. Any suitable bacterialorganism finds use in the present disclosure, including but not limitedto Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter,Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium,Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter,Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia,Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium,Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter,Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus,Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium,Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus,Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia,Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus,Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella,Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula,Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella,Yersinia and Zymomonas. In some embodiments, the host cell is a speciesof Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium,Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium,Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus,Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella,Streptococcus, Streptomyces, or Zymomonas. In some embodiments, thebacterial host strain is non-pathogenic to humans. In some embodimentsthe bacterial host strain is an industrial strain. Numerous bacterialindustrial strains are known and suitable in the present disclosure. Insome embodiments of the present disclosure, the bacterial host cell isan Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A.rubi). In some embodiments of the present disclosure, the bacterial hostcell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A.globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A.paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A.ureafaciens). In some embodiments of the present disclosure, thebacterial host cell is a Bacillus species (e.g., B. thuringensis, B.anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B.pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius,B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, andB. amyloliquefaciens). In some embodiments, the host cell is anindustrial Bacillus strain including but not limited to B. subtilis, B.pumilus, B. licheniformis, B. megaterium, B. clausii, B.stearothermophilus, or B. amyloliquefaciens. In some embodiments, theBacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B.stearothermophilus, and/or B. amyloliquefaciens. In some embodiments,the bacterial host cell is a Clostridium species (e.g., C.acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum,C. perfringens, and C. beijerinckii). In some embodiments, the bacterialhost cell is a Corynebacterium species (e.g., C. glutamicum and C.acetoacidophilum). In some embodiments the bacterial host cell is anEscherichia species (e.g., E. coli). In some embodiments, the host cellis Escherichia coli W3110. In some embodiments, the bacterial host cellis an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas, E.herbicola, E. punctata, and E. terreus). In some embodiments, thebacterial host cell is a Pantoea species (e.g., P. citrea, and P.agglomerans). In some embodiments the bacterial host cell is aPseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, andP. sp. D-01 10). In some embodiments, the bacterial host cell is aStreptococcus species (e.g., S. equisimiles, S. pyogenes, and S.uberis). In some embodiments, the bacterial host cell is a Streptomycesspecies (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S.coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, andS. lividans). In some embodiments, the bacterial host cell is aZymomonas species (e.g., Z. mobilis, and Z. lipolytica).

Many prokaryotic and eukaryotic strains that find use in the presentdisclosure are readily available to the public from a number of culturecollections such as American Type Culture Collection (ATCC), DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH (DSM), CentraalbureauVoor Schimmelcultures (CBS), and Agricultural Research Service PatentCulture Collection, Northern Regional Research Center (NRRL).

In some embodiments, host cells are genetically modified to havecharacteristics that improve protein secretion, protein stability and/orother properties desirable for expression and/or secretion of a protein.Genetic modification can be achieved by genetic engineering techniquesand/or classical microbiological techniques (e.g., chemical or UVmutagenesis and subsequent selection). Indeed, in some embodiments,combinations of recombinant modification and classical selectiontechniques are used to produce the host cells. Using recombinanttechnology, nucleic acid molecules can be introduced, deleted, inhibitedor modified, in a manner that results in increased yields oftransaminase variant(s) within the host cell and/or in the culturemedium. For example, knockout of AlpI function results in a cell that isprotease deficient, and knockout of pyr5 function results in a cell witha pyrimidine deficient phenotype. In one genetic engineering approach,homologous recombination is used to induce targeted gene modificationsby specifically targeting a gene in vivo to suppress expression of theencoded protein. In alternative approaches, siRNA, antisense and/orribozyme technology find use in inhibiting gene expression. A variety ofmethods are known in the art for reducing expression of protein incells, including, but not limited to deletion of all or part of the geneencoding the protein and site-specific mutagenesis to disrupt expressionor activity of the gene product. (See e.g., Chaveroche et al., Nucl.Acids Res., 28:22 e97 [2000]; Cho et al., Mol. Plant Mic. Interact.,19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett., 30:1811-1817[2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352 [2004]; and Youet al., Arch. Micriobiol., 191:615-622 [2009], all of which areincorporated by reference herein). Random mutagenesis, followed byscreening for desired mutations also finds use (See e.g., Combier etal., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon et al., Eukary.Cell 2:247-55 [2003], both of which are incorporated by reference).

Introduction of a vector or DNA construct into a host cell can beaccomplished using any suitable method known in the art, including butnot limited to calcium phosphate transfection, DEAE-dextran mediatedtransfection, PEG-mediated transformation, electroporation, or othercommon techniques known in the art. In some embodiments, the Escherichiacoli expression vector pCK100900i (See US Pat. Appln. Publn.2006/0195947, which is hereby incorporated by reference herein) finduse.

In some embodiments, the engineered host cells (i.e., “recombinant hostcells”) of the present disclosure are cultured in conventional nutrientmedia modified as appropriate for activating promoters, selectingtransformants, or amplifying the transaminase polynucleotide. Cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and are well-known tothose skilled in the art. As noted, many standard references and textsare available for the culture and production of many cells, includingcells of bacterial, plant, animal (especially mammalian) andarchebacterial origin.

In some embodiments, cells expressing the variant transaminasepolypeptides of the disclosure are grown under batch or continuousfermentations conditions. Classical “batch fermentation” is a closedsystem, wherein the compositions of the medium are set at the beginningof the fermentation and is not subject to artificial alternations duringthe fermentation. A variation of the batch system is a “fed-batchfermentation” which also finds use in the present disclosure. In thisvariation, the substrate is added in increments as the fermentationprogresses. Fed-batch systems are useful when catabolite repression islikely to inhibit the metabolism of the cells and where it is desirableto have limited amounts of substrate in the medium. Batch and fed-batchfermentations are common and well known in the art. “Continuousfermentation” is an open system where a defined fermentation medium isadded continuously to a bioreactor and an equal amount of conditionedmedium is removed simultaneously for processing. Continuous fermentationgenerally maintains the cultures at a constant high density where cellsare primarily in log phase growth. Continuous fermentation systemsstrive to maintain steady state growth conditions. Methods formodulating nutrients and growth factors for continuous fermentationprocesses as well as techniques for maximizing the rate of productformation are well known in the art of industrial microbiology.

In some embodiments of the present disclosure, cell-freetranscription/translation systems find use in producing varianttransaminase(s). Several systems are commercially available, and themethods are well-known to those skilled in the art.

The present disclosure provides methods of making variant transaminasepolypeptides or biologically active fragments thereof. In someembodiments, the method comprises: providing a host cell transformedwith a polynucleotide encoding an amino acid sequence that comprises atleast about 70% (or at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%) sequenceidentity to SEQ ID NO: 2, 4, 8, 366, and/or 650, and comprising at leastone mutation as provided herein; culturing the transformed host cell ina culture medium under conditions in which the host cell expresses theencoded variant transaminase polypeptide; and optionally recovering orisolating the expressed variant transaminase polypeptide, and/orrecovering or isolating the culture medium containing the expressedvariant transaminase polypeptide. In some embodiments, the methodsfurther provide optionally lysing the transformed host cells afterexpressing the encoded transaminase polypeptide and optionallyrecovering and/or isolating the expressed variant transaminasepolypeptide from the cell lysate. The present disclosure furtherprovides methods of making a variant transaminase polypeptide comprisingcultivating a host cell transformed with a variant transaminasepolypeptide under conditions suitable for the production of the varianttransaminase polypeptide and recovering the variant transaminasepolypeptide. Typically, recovery or isolation of the transaminasepolypeptide is from the host cell culture medium, the host cell or both,using protein recovery techniques that are well known in the art,including those described herein. In some embodiments, host cells areharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including, but not limited to freeze-thawcycling, sonication, mechanical disruption, and/or use of cell lysingagents, as well as many other suitable methods well known to thoseskilled in the art.

Engineered transaminase enzymes produced by a host cell can be recoveredfrom the cells and/or the culture medium using any one or more of thetechniques known in the art for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™(Sigma-Aldrich). Thus, in some embodiments, the resulting polypeptide isrecovered/isolated and optionally purified by any of a number of methodsknown in the art. For example, in some embodiments, the polypeptide isisolated from the nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, chromatography (e.g., ion exchange, affinity,hydrophobic interaction, chromatofocusing, and size exclusion), orprecipitation. In some embodiments, protein refolding steps are used, asdesired, in completing the configuration of the mature protein. Inaddition, in some embodiments, high performance liquid chromatography(HPLC) is employed in the final purification steps. For example, in someembodiments, methods known in the art, find use in the presentdisclosure (See e.g., Parry et al., Biochem. J., 353:117 [2001]; andHong et al., Appl. Microbiol. Biotechnol., 73:1331 [2007], both of whichare incorporated herein by reference). Indeed, any suitable purificationmethods known in the art find use in the present disclosure.

Chromatographic techniques for isolation of the transaminase polypeptideinclude, but are not limited to reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,are known to those skilled in the art.

In some embodiments, affinity techniques find use in isolating theimproved transaminase enzymes. For affinity chromatography purification,any antibody which specifically binds the transaminase polypeptide maybe used. For the production of antibodies, various host animals,including but not limited to rabbits, mice, rats, etc., may be immunizedby injection with the transaminase. The transaminase polypeptide may beattached to a suitable carrier, such as BSA, by means of a side chainfunctional group or linkers attached to a side chain functional group.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(Bacillus Calmette Guerin) and Corynebacterium parvum.

In some embodiments, the transaminase variants are prepared and used inthe form of cells expressing the enzymes, as crude extracts, or asisolated or purified preparations. In some embodiments, the transaminasevariants are prepared as lyophilisates, in powder form (e.g., acetonepowders), or prepared as enzyme solutions. In some embodiments, thetransaminase variants are in the form of substantially purepreparations.

In some embodiments, the transaminase polypeptides are attached to anysuitable solid substrate. Solid substrates include but are not limitedto a solid phase, surface, and/or membrane. Solid supports include, butare not limited to organic polymers such as polystyrene, polyethylene,polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide,as well as co-polymers and grafts thereof. A solid support can also beinorganic, such as glass, silica, controlled pore glass (CPG), reversephase silica or metal, such as gold or platinum. The configuration ofthe substrate can be in the form of beads, spheres, particles, granules,a gel, a membrane or a surface. Surfaces can be planar, substantiallyplanar, or non-planar. Solid supports can be porous or non-porous, andcan have swelling or non-swelling characteristics. A solid support canbe configured in the form of a well, depression, or other container,vessel, feature, or location. A plurality of supports can be configuredon an array at various locations, addressable for robotic delivery ofreagents, or by detection methods and/or instruments.

In some embodiments, immunological methods are used to purifytransaminase variants. In one approach, antibody raised against avariant transaminase polypeptide (e.g., against a polypeptide comprisingan engineered transaminase variant provided herein, including, but notlimited to SEQ ID NO: 2, 4, 8, 366, and/or 650, and/or an immunogenicfragment thereof) using conventional methods is immobilized on beads,mixed with cell culture media under conditions in which the varianttransaminase is bound, and precipitated. In a related approach,immunochromatography finds use.

In some embodiments, the variant transaminases are expressed as a fusionprotein including a non-enzyme portion. In some embodiments, the varianttransaminase sequence is fused to a purification facilitating domain. Asused herein, the term “purification facilitating domain” refers to adomain that mediates purification of the polypeptide to which it isfused. Suitable purification domains include, but are not limited tometal chelating peptides, histidine-tryptophan modules that allowpurification on immobilized metals, a sequence which binds glutathione(e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitopederived from the influenza hemagglutinin protein; See e.g., Wilson etal., Cell 37:767 [1984]), maltose binding protein sequences, the FLAGepitope utilized in the FLAGS extension/affinity purification system(e.g., the system available from Immunex Corp), and the like. Oneexpression vector contemplated for use in the compositions and methodsdescribed herein provides for expression of a fusion protein comprisinga polypeptide of the disclosure fused to a polyhistidine regionseparated by an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography; See e.g., Porath et al., Prot. Exp. Purif., 3:263-281[1992]) while the enterokinase cleavage site provides a means forseparating the variant transaminase polypeptide from the fusion protein.pGEX vectors (Promega) may also be used to express foreign polypeptidesas fusion proteins with glutathione S-transferase (GST). In general,such fusion proteins are soluble and can easily be purified from lysedcells by adsorption to ligand-agarose beads (e.g., glutathione-agarosein the case of GST-fusions) followed by elution in the presence of freeligand.

Methods of Using the Engineered Transaminase Enzymes

In another aspect, the engineered transaminase polypeptides disclosedherein can be used in a process for the conversion of the substratecompound (1), or structural analogs thereof, to the product of compound(2) or the corresponding structural analog.

As described herein, and illustrated in the Examples, the presentdisclosure contemplates ranges of suitable reaction conditions that canbe used in the processes herein, including but not limited to ranges ofpH, temperature, buffer, solvent system, substrate loading, mixture ofsubstrate compound stereoisomers, polypeptide loading, cofactor loading,pressure, and reaction time. Further suitable reaction conditions forcarrying out the process for biocatalytic conversion of substratecompounds to product compounds using an engineered transaminasepolypeptide described herein can be readily optimized by routineexperimentation that includes, but is not limited to, contacting theengineered transaminase polypeptide and substrate compound underexperimental reaction conditions of concentration, pH, temperature,solvent conditions, and detecting the product compound, for example,using the methods described in the Examples provided herein.

As described above, the engineered polypeptides having transaminaseactivity for use in the processes of the present disclosure generallycomprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identityto a reference amino acid sequence selected from any one of theeven-numbered sequences of SEQ ID NO: 6-936, and an engineeredtransaminase polypeptide comprising an amino acid sequence that has (a)has one or more amino acid residue differences as compared to areference sequence (e.g., SEQ ID NO: 2, 4, 8, 366, and/or 650). In someembodiments, the polynucleotide capable of hybridizing under highlystringent conditions encodes a transaminase polypeptide that has thepercent identity described above and one or more residue differences ascompared to a reference sequence (e.g., SEQ ID NO: 2, 4, 8, 366, and/or650).

Substrate compound in the reaction mixtures can be varied, taking intoconsideration, for example, the desired amount of product compound, theeffect of substrate concentration on enzyme activity, stability ofenzyme under reaction conditions, and the percent conversion ofsubstrate to product. In some embodiments of the method, the suitablereaction conditions comprise a substrate compound loading of at leastabout 0.5 to about 200 g/L, 1 to about 200 g/L, 5 to about 150 g/L,about 10 to about 100 g/L, or about 50 to about 100 g/L. In someembodiments, the suitable reaction conditions comprise a substratecompound loading of at least about 0.5 g/L, at least about 1 g/L, atleast about 5 g/L, at least about 10 g/L, at least about 15 g/L, atleast about 20 g/L, at least about 30 g/L, at least about 50 g/L, atleast about 75 g/L, at least about 100 g/L, at least about 150 g/L or atleast about 200 g/L, or even greater. The values for substrate loadingsprovided herein are based on the molecular weight of compound (1),however it also contemplated that the equivalent molar amounts ofvarious hydrates and salts of compound (1) also can be used in theprocess.

In the processes describes herein, the engineered transaminasepolypeptide uses an amino donor to form the product compounds. In someembodiments, the amino donor in the reaction condition comprises acompound selected from isopropylamine (also referred to herein as “IPM”)or any other suitable amino donor for the reaction of interest. In someembodiments, the amino donor is IPM. In some embodiments, the suitablereaction conditions comprise the amino donor, in particular IPM, presentat a concentration of at least about 0.1 to about 3.0 M, 0.2 to about2.5 M, about 0.5 to about 2 M or about 1 to about 2 M. In someembodiments, the amino donor is present at a concentration of about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.5, 2, 2.5 or 3 M.

Suitable reaction conditions for the processes also typically comprisethe presence of a cofactor in the reaction mixture. Because theengineered transaminases typically use members of the vitamin B₆ family,the reaction condition can comprise a cofactor selectedfrom-pyridoxal-5′-phosphate (also known as pyridoxal-phosphate, PLP,P5P), pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and theirphosphorylated counterparts; pyridoxine phosphate (PNP), andpyridoxamine phosphate (PMP). In some embodiments, the suitable reactionconditions can comprise the presence of a cofactor selected from PLP,PN, PL, PM, PNP, and PMP, at a concentration of about 0.1 g/L to about10 g/L, about 0.2 g/L to about 5 g/L, about 0.5 g/L to about 2.5 g/L. Insome embodiments, the cofactor is PLP. Accordingly, in some embodiments,the suitable reaction conditions can comprise the presence of thecofactor, PLP, at a concentration of about 0.1 g/L to about 10 g/L,about 0.2 g/L to about 5 g/L, about 0.5 g/L to about 2.5 g/L. In someembodiments, the reaction conditions comprise a PLP concentration ofabout 10 g/L or less, about 5 g/L or less, about 2.5 g/L or less, about1.0 g/L or less, about 0.5 g/L or less, or about 0.2 g/L or less.

In some embodiments of the process (e.g., where whole cells or lysatesare used), the cofactor is present naturally in the cell extract anddoes not need to be supplemented. In some embodiments of the process(e.g., using partially purified, or purified transaminase enzyme), theprocess can further comprise a step of adding cofactor to the enzymereaction mixture. In some embodiments, the cofactor is added either atthe beginning of the reaction and/or additional cofactor is added duringthe reaction.

During the course of the transamination reactions, the pH of thereaction mixture may change. The pH of the reaction mixture may bemaintained at a desired pH or within a desired pH range. This may bedone by the addition of an acid or a base, before and/or during thecourse of the reaction. Alternatively, the pH may be controlled by usinga buffer. Accordingly, in some embodiments, the reaction conditioncomprises a buffer. Suitable buffers to maintain desired pH ranges areknown in the art and include, by way of example and not limitation,borate, carbonate, phosphate, triethanolamine (TEA) buffer, and thelike. In some embodiments, the buffer is TEA. In some embodiments of theprocess, the suitable reaction conditions comprise a buffer solution ofTEA, where the TEA concentration is from about 0.01 to about 0.4 M, 0.05to about 0.4 M, 0.1 to about 0.3 M, or about 0.1 to about 0.2 M. In someembodiments, the reaction condition comprises a TEA concentration ofabout 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12, 0.14, 0.16, 0.18,0.2, 0.3, or 0.4 M. In some embodiments, the reaction conditionscomprise water as a suitable solvent with no buffer present.

In some embodiments of the process, the reaction conditions can comprisea suitable pH. As noted above, the desired pH or desired pH range can bemaintained by use of an acid or base, an appropriate buffer, or acombination of buffering and acid or base addition. The pH of thereaction mixture can be controlled before and/or during the course ofthe reaction. In some embodiments, the suitable reaction conditionscomprise a solution pH of about 8 to about 12.5, a pH of about 8 toabout 12, a pH of about 9.0 to about 11.5, or a pH of about 9.5 to about11.0. In some embodiments, the reaction conditions comprise a solutionpH of about 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 or 12.5.

In some embodiments of the processes herein, a suitable temperature canbe used for the reaction conditions, for example, taking intoconsideration the increase in reaction rate at higher temperatures, theactivity of the enzyme for sufficient duration of the reaction, and asfurther described below. For example, the engineered polypeptides of thepresent disclosure have increased stability relative to naturallyoccurring transaminase polypeptide, which allows the engineeredpolypeptides of the present disclosure to be used at higher temperaturesfor increased conversion rates and improved substrate solubilitycharacteristics for the reaction. Accordingly, in some embodiments, thesuitable reaction conditions comprise a temperature of about 10° C. toabout 70° C., about 10° C. to about 65° C., about 15° C. to about 60°C., about 20° C. to about 60° C., about 20° C. to about 55° C., about30° C. to about 55° C., or about 40° C. to about 50° C. In someembodiments, the suitable reaction conditions comprise a temperature ofabout 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C.,50° C., 55° C., 60° C., 65° C., or 70° C. In some embodiments, thetemperature during the enzymatic reaction can be maintained at atemperature throughout the course of the reaction. In some embodiments,the temperature during the enzymatic reaction can be adjusted over atemperature profile during the course of the reaction.

In some embodiments of the process, the suitable reaction conditions canfurther comprise the presence of the reduced cofactor, nicotinamideadenine dinucleotide (NADH), which can act to limit the inactivation ofthe transaminase enzyme (See e.g., van Ophem et al., Biochem.,37(9):2879-88 [1998]). In such embodiments where NADH is present, acofactor regeneration system, such as glucose dehydrogenase (GDH) andglucose or formate dehydrogenase and formate can be used to regeneratethe NADH in the reaction medium.

The processes using the engineered transaminases are generally carriedout in a solvent. Suitable solvents include water, aqueous buffersolutions, organic solvents, and/or co-solvent systems, which generallycomprise aqueous solvents and organic solvents. The aqueous solvent(water or aqueous co-solvent system) may be pH-buffered or unbuffered.In some embodiments, the processes using the engineered transaminasepolypeptides are generally carried out in an aqueous co-solvent systemcomprising an organic solvent (e.g., ethanol, isopropanol (IPA),dimethyl sulfoxide (DMSO), ethyl acetate, butyl acetate, 1-octanol,heptane, octane, methyl t-butyl ether (MTBE), toluene, and the like),ionic liquids (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, and the like). Theorganic solvent component of an aqueous co-solvent system may bemiscible with the aqueous component, providing a single liquid phase, ormay be partly miscible or immiscible with the aqueous component,providing two liquid phases. Exemplary aqueous co-solvent systemscomprises water and one or more organic solvent. In general, an organicsolvent component of an aqueous co-solvent system is selected such thatit does not completely inactivate the transaminase enzyme. Appropriateco-solvent systems can be readily identified by measuring the enzymaticactivity of the specified engineered transaminase enzyme with a definedsubstrate of interest in the candidate solvent system, utilizing anenzyme activity assay, such as those described herein. In someembodiments of the process, the suitable reaction conditions comprise anaqueous co-solvent comprising DMSO at a concentration of about 1% toabout 80% (v/v), about 1 to about 70% (v/v), about 2% to about 60%(v/v), about 5% to about 40% (v/v), 10% to about 40% (v/v), 10% to about30% (v/v), or about 10% to about 20% (v/v). In some embodiments of theprocess, the suitable reaction conditions comprise an aqueous co-solventcomprising DMSO at a concentration of at least about 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%(v/v).

The suitable reaction conditions can comprise a combination of reactionparameters that provide for the biocatalytic conversion of the substratecompounds to its corresponding product compounds. For example, in someembodiments, the preparation of compound (2) can be carried out whereinthe suitable reaction conditions comprise: (a) substrate loading ofabout 10 to 300 g/L of substrate compound (e.g., 50 g/L or 200 g/L ofcompound (1)); (b) of about 0.5 g/L to 60 g/L engineered polypeptide;(c) IPM concentration of about 0.5 to 2 M; (d) PLP cofactorconcentration of about 0.1 to 1 g/L; (e) DMSO concentration of about 0%(v/v) to about 20% (v/v); (f) pH of about 8.5 to 11.5; and (g)temperature of about 45° C. to 65° C.

In some embodiments, the suitable reaction conditions comprise: (a)about 100 g/L of substrate compound (e.g., compound (1)); (b) about 1g/L engineered polypeptide; (c) about 1 M isopropylamine (IPM); (d)about 0.5 g/L pyridoxal phosphate (PLP); (e) about pH 9; and (g) about50° C.

Further exemplary reaction conditions include the assay conditionsprovided in the Examples. In carrying out the transamination reactionsdescribed herein, the engineered transaminase polypeptide may be addedto the reaction mixture in the partially purified or purified enzyme,whole cells transformed with gene(s) encoding the enzyme, and/or as cellextracts and/or lysates of such cells. Whole cells transformed withgene(s) encoding the engineered transaminase enzyme or cell extracts,lysates thereof, and isolated enzymes may be employed in a variety ofdifferent forms, including solid (e.g., lyophilized, spray-dried, andthe like) or semisolid (e.g., a crude paste). The cell extracts or celllysates may be partially purified by precipitation (e.g., ammoniumsulfate, polyethyleneimine, heat treatment or the like), followed by adesalting procedure (e.g., ultrafiltration, dialysis, and the like)prior to lyophilization. Any of the enzyme preparations may bestabilized by crosslinking using known crosslinking agents, such as, forexample, glutaraldehyde, or immobilized to a solid phase material (e.g.,resins, beads such as chitosan, Eupergit C, SEPABEADs, and the like).

In some embodiments of the transamination reactions described herein,the reaction is carried out under the suitable reaction conditionsdescribed herein, wherein the engineered transaminase polypeptide isimmobilized to a solid support. Solid supports useful for immobilizingthe engineered transaminases for carrying out the transaminationreactions include but are not limited to beads or resins comprisingpolymethacrylate with epoxide functional groups, polymethacrylate withamino epoxide functional groups, styrene/DVB copolymer orpolymethacrylate with octadecyl functional groups. Exemplary solidsupports include, but are not limited to, chitosan beads, Eupergit C,and SEPABEADs (Mitsubishi), including the following different types ofSEPABEAD: EC-EP, EC-HFA/S, EXA252, EXE119 and EXE120.

In some embodiments where the engineered polypeptide can be expressed inthe form of a secreted polypeptide, the culture medium containing thesecreted polypeptides can be used in the process herein.

In some embodiments, solid reactants (e.g., enzyme, salts, etc.) may beprovided to the reaction in a variety of different forms, includingpowder (e.g., lyophilized, spray dried, and the like), solution,emulsion, suspension, and the like. The reactants can be readilylyophilized or spray dried using methods and equipment that are known tothose having ordinary skill in the art. For example, the proteinsolution can be frozen at −80° C. in small aliquots, then added to apre-chilled lyophilization chamber, followed by the application of avacuum.

In some embodiments, the order of addition of reactants is not critical.The reactants may be added together at the same time to a solvent (e.g.,monophasic solvent, biphasic aqueous co-solvent system, and the like),or alternatively, some of the reactants may be added separately, andsome together at different time points. For example, the cofactor,transaminase, and transaminase substrate may be added first to thesolvent. For improved mixing efficiency when an aqueous co-solventsystem is used, the transaminase, and cofactor may be added and mixedinto the aqueous phase first. The organic phase may then be added andmixed in, followed by addition of the transaminase substrate.Alternatively, the transaminase substrate may be premixed in the organicphase, prior to addition to the aqueous phase.

In some embodiments, where the choice of the amino donor results in acarbonyl by-product that has a vapor pressure higher than water (e.g., alow boiling co-product such as a volatile organic carbonyl compound),the carbonyl by-product can be removed by sparging the reaction solutionwith a non-reactive gas or by applying a vacuum to lower the reactionpressure and removing the carbonyl by-product present in the gas phase.A non-reactive gas is any gas that does not react with the reactioncomponents. Various non-reactive gases include nitrogen and noble gases(e.g., inert gases). In some embodiments, the non-reactive gas isnitrogen gas. In some embodiments, the amino donor used in the processis isopropylamine (IPM), which forms the carbonyl by-product acetoneupon transfer of the amino group to the amino group acceptor. Theacetone can be removed by sparging with nitrogen gas or applying avacuum to the reaction solution and removing the acetone from the gasphase by an acetone trap, such as a condenser or other cold trap.Alternatively, the acetone can be removed by reduction to isopropanolusing a ketoreductase.

In some embodiments of the process where the carbonyl by-product isremoved, the corresponding amino group donor can be added during thetransamination reaction to replenish the amino group donor and/ormaintain the pH of the reaction. Replenishing the amino group donor alsoshifts the equilibrium towards product formation, thereby increasing theconversion of substrate to product. Thus, in some embodiments where theamino group donor is IPM and the acetone product is removed in situ, theprocess can further comprise a step of adding IPM to the reactionsolution to replenish the amino group donor lost during the acetoneremoval and to maintain the pH of the reaction (e.g., at about 8.5 toabout pH 11.5).

In some embodiments, it is also contemplated that the process comprisingthe biocatalytic conversion of amine acceptor substrate compounds tochiral amine product compounds using transaminase polypeptides of thepresent disclosure can further comprise steps of formation ofpharmaceutically acceptable salts or acids, pharmaceutically acceptableformulations, product work-up, extraction, isolation, purification,and/or crystallization, each of which can be carried out under a rangeof conditions.

In some embodiments, the processes using the engineered polypeptidesdisclosed herein can be carried out wherein the amino group donor isselected from isopropylamine, alanine, 3-aminobutyric acid, ormethylbenzylamine. In some embodiments, the amino group donor isisopropylamine.

Methods, techniques, and protocols for extracting, isolating, forming asalt of, purifying, and/or crystallizing aminated product compounds orcyclized compounds from biocatalytic reaction mixtures produced by theabove disclosed processes are known to the ordinary artisan and/oraccessed through routine experimentation. Additionally, illustrativemethods are provided in the Examples below.

Various features and embodiments of the invention are illustrated in thefollowing representative examples, which are intended to beillustrative, and not limiting.

EXPERIMENTAL

Various features and embodiments of the invention are illustrated in thefollowing representative examples, which are intended to beillustrative, and not limiting.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and μg (micrograms); L and l (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);° C. (degrees Centigrade); RT (room temperature); CDS (coding sequence);DNA (deoxyribonucleic acid); RNA (ribonucleic acid); aa (amino acid); TB(Terrific Broth; 12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/Lglycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO₄); LB (Luriabroth); CAM (chloramphenicol); PMBS (polymyxin B sulfate); IPTG(isopropyl thiogalactoside); ATA (omega-transaminase); TFA(trifluoroacetic acid); TEoA (triethanolamine); borate (sodiumtetraborate decahydrate); acetonitrile (MeCN); dimethylsulfoxide (DMSO);HPLC (high performance liquid chromatography); FIOP (fold improvementover positive control); HTP (high throughput); MWD (multiple wavelengthdetector); UV (ultraviolet); Codexis (Codexis, Inc., Redwood City, CA);Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Millipore (Millipore,Corp., Billerica MA); Difco (Difco Laboratories, BD Diagnostic Systems,Detroit, MI); Daicel (Daicel, West Chester, PA); Genetix (Genetix USA,Inc., Beaverton, OR); Molecular Devices (Molecular Devices, LLC,Sunnyvale, CA); Applied Biosystems (Applied Biosystems, part of LifeTechnologies, Corp., Grand Island, NY), Agilent (Agilent Technologies,Inc., Santa Clara, CA); Thermo Scientific (part of Thermo FisherScientific, Waltham, MA); (Infors; Infors-HT, Bottmingen/Basel,Switzerland); Corning (Corning, Inc., Palo Alto, CA); and Bio-Rad(Bio-Rad Laboratories, Hercules, CA); Microfluidics (MicrofluidicsCorp., Newton, MA); Waters (Waters Corp., Milford, MA).

Example 1 Production of Engineered Polypeptides in pCK110900

The polynucleotide (SEQ ID NO: 1) encoding the polypeptide from Vibriofluvialis having transaminase activity (SEQ ID NO: 2) was cloned into apCK110900 vector system (See e.g., U.S. Pat. No. 9,714,437, which ishereby incorporated by reference in its entirety) and subsequentlyexpressed in E. coli W3110fhuA under the control of the lac promoter.

In a 96-well format, single colonies were picked and grown in 190 μL LBmedia containing 1% glucose and 30 μg/mL chloramphenicol (CAM), at 30°C., 200 rpm, and 85% humidity. Following overnight growth, 20 μL of thegrown cultures were transferred into a deep-well plate containing 380 μLof TB with 30 μg/mL CAM. The cultures were grown at 30° C., 250 rpm,with 85% humidity for approximately 2.5 hours. When the optical density(OD₆₀₀) of the cultures reached 0.4-0.6, expression of the transaminasegene was induced by addition of IPTG to a final concentration of 1 mM.Following induction, growth was continued for 18-20 hours at 30° C., 250rpm with 85% humidity. Cells were harvested by centrifugation at 4000rpm at 4° C. for 10 minutes, and the media was discarded. The cellpellets were stored at −80° C. until ready for use. Prior to performingthe assay, cell pellets were resuspended in 400 μL of lysis buffercontaining 50 mM triethanolamine-HCl, pH 7.5, with 1 g/L PLP, 1 g/Llysozyme, and 0.5 g/L PMBS. The plates were agitated with medium-speedshaking for 2 hours on a microtiter plate shaker at room temperature.The plates were then centrifuged at 4000 rpm for 15 minutes at 4° C.,and the clarified supernatants were used in the HTP assay reactiondescribed below.

Shake-flask procedures can be used to generate engineered transaminasepolypeptide shake-flask powders (SFP), which are useful for secondaryscreening assays and/or use in the biocatalytic processes describedherein. Shake flask powder preparation of enzymes provides a morepurified preparation (e.g., up to 30% of total protein) of theengineered enzyme, as compared to the cell lysate used in HTP assays andalso allows for the use of more concentrated enzyme solutions. To startthe cultures, a single colony of E. coli containing a plasmid encodingan engineered polypeptide of interest was inoculated into 5 mL LB with30 μg/mL CAM and 1% glucose. The culture was grown overnight (at least16 hours) in an incubator at 37° C., with shaking at 250 rpm. The grownculture was then added to 250 mL of TB with 30 μg/mL CAM, in a 1Lshake-flask. The 250 mL culture was grown at 30° C. at 250 rpm for 3.5hours until the OD₆₀₀ reached 0.6-0.8. Expression of the transaminasegene was induced by addition of IPTG to a final concentration of 1 mM,and growth was continued for an additional 18-20 hours. Cells wereharvested by transferring the culture into a pre-weighed centrifugebottle, then centrifuged at 4000 rpm for 20 minutes at 4° C. Thesupernatant was discarded, and the remaining cell pellet was weighed. Insome embodiments, the cells were stored at −80° C. until ready to use.For lysis, the cell pellet was resuspended in 6 mL/g wet cell weight of25 mM triethanolamine-HCl buffer, pH 7.5 and lysed using a 110LMICROFLUIDIZER© processor system (Microfluidics). Cell debris wasremoved by centrifugation at 10,000 rpm for 60 minutes at 4° C. Theclarified lysate was collected, frozen at −80° C., and then lyophilized,using standard methods known in the art. Lyophilization of frozenclarified lysate provides a dry shake-flask powder comprising crudeengineered polypeptide.

Example 2 Evolution and Screening of Engineered Polypeptides Derivedfrom SEQ ID NO: 4 for Improved Production of Compound (2)

The engineered polynucleotide (SEQ ID NO: 3) encoding the polypeptidewith transaminase activity of SEQ ID NO: 4 was used to generate theengineered polypeptides of Table 2-1. These polypeptides displayedimproved transaminase activity under the desired conditions e.g., theimprovement in the formation of the amine product, compound (2), fromthe ketone substrate, compound (1), as compared to the startingpolypeptide. The engineered polypeptides, having the amino acidsequences of even-numbered sequence identifiers were generated from the“backbone” amino acid sequence of SEQ ID NO: 4 as described below,together with the HTP assay and analytical methods described in Tables6-1 and 6-2.

Directed evolution began with the polynucleotide set forth in SEQ ID NO:3. Libraries of engineered polypeptides were generated using variouswell-known techniques (e.g., saturation mutagenesis, recombination ofpreviously identified beneficial amino acid differences) and screenedusing HTP assay and analysis methods that measured the polypeptides'ability to produce compound (2).

The enzyme assay was carried out in 96-well shallow-well (300 μLvolume/well) plates, in 100 μL total reaction volume/well. The reactioncontained 55 μL of 64-fold diluted ATA lysate (diluted in 50 mMtriethanolamine+1 g/L PLP, pH 7.5), 25 μL of 4 M isopropylamine-HCl, pH9, and 20 μL of 100 g/L ketone (1) dissolved in DMSO. The reaction platewas heat-sealed and shaken at 600 rpm at 50° C. for 22 hours.

After overnight incubation (˜22 hours), 100 μL/well of 50% formic acidin acetonitrile was added to the reaction plate and mixed well. Theplates were sealed and centrifuged at 4000 rpm for 10 min. A 20 μL/wellaliquot was removed from the quenched plate and diluted into 180 μL of1:1 acetonitrile:water and analyzed by HPLC to determine activity andselectivity as described in Tables 6-1 and 6-2, respectively.

Hit variants were grown in 250 mL shake flasks as described in Example 1to generate lyophilized enzyme powders. The activity of the enzymepowders were evaluated at 0-10 g/L of the ATA shake flask powder, 20-100g/L ketone, 1 M IPM (4 M pH 9 stock), 0-40% cosolvent (DMSO ormethanol), pH˜9, 50° C., for 20-24 hours, using a similar assay asdescribed above and the hit variants are listed in Table 2-2.

TABLE 2-1 Transaminase Activity in the Production of Compound (2)Relative to SEQ ID NO: 4 SEQ Percent Conversion ID Fold Improvement NO:Amino Acid Differences (Relative to (nt/aa) (Relative to SEQ ID NO: 4)SEQ ID NO: 4)¹ 7/8 K163F; N286S; I314R; E316W; A323T; +++ C414V; P416A 9/10 N286S; I314R; A323T; A383V +++ 11/12 K163F; N286S; I314R; A323T;D394G +++ 13/14 N286S; E316W; A323T; A383V; +++ C414V; P416A 15/16 A74T;G81S; N286S; E316W; A323T; +++ A383V 17/18 A74T; N286S; E316W; A323T +++5/6 T408F +++ 19/20 E315G +++ 21/22 N286S; E316W; D394G; C414V; P416A+++ 23/24 N286S; A323T; A383V; C414V +++ 25/26 K163F; A222V; N286S;E316W; A323T; +++ A383V; D394G 27/28 N286S; A323T ++ 29/30 K163F; N286S;C414V ++ 31/32 N286S; A323T; P416A ++ 33/34 N286S ++ 35/36 N286S; E316W;A383V; D394G ++ 37/38 A74T; K163F; I314R; A323T; A383V; ++ C414V; P416A39/40 N286S; I314R; D394G ++ 41/42 I314R; A323T; A383V; D394G ++ 43/44K163F; I314R; C414V ++ 45/46 K163F; N286S ++ 47/48 N286S; A383V ++ 49/50K163F; I314R; A383V ++ 51/52 A74T; I314R; A323T; A383V; D394G; ++ C414V53/54 K163F; I314R; A323T; D394G ++ 55/56 K163F; N286S; E316W; A323T; ++D394G; P416A 57/58 K163F; A323T ++ 59/60 A323S ++ 61/62 L417S ++ 63/64I314R; E316W; A323T ++ 65/66 T408W ++ 67/68 A323C ++ 69/70 A323T ++71/72 A74T; K163F; I314R; E316W; A323T; ++ D394G 73/74 F85V; N286S;A323T ++ 75/76 A323T; D394G ++ 77/78 E316F ++ 79/80 I314R; E316W; A323T;D394G ++ 81/82 A323T; A383V + 83/84 N286S; P416A + 85/86 I259V + 87/88A74T; K163F; N286S; E316W; A383V; + D394G; P416A 89/90 A74T; N286S +91/92 F85V; S86A; K163F; I314R; A323T; + D394G; C414V 93/94 H88R; N286S;E316W; A323T; A383V; + C414V; P416A 95/96 I314R; E316W; D394G + 97/98H88R; K163F; N286S; A383V +  99/100 I314R; E316W + 101/102 A74T; K163F;I314R; E316W + 103/104 L56A; N286S; I314R; E316W; A323T; + V422A 105/106I314R; E316W; A323T; A383V; V422A + 107/108 D21H + 109/110 A323T; A383V;D394G; C414V; P416A + 111/112 L56A; N286S; A383V + 113/114 K163L +115/116 P23S; L417S + 117/118 K163F; A383V + 119/120 E316G + 121/122V422T + 123/124 G395D; L417S + 125/126 E316N + 127/128 R146H; L417S +129/130 K163M + 131/132 K163F + 133/134 A74T; H88R; N286S; E316W;A323T; + A383V 135/136 P164Q + 137/138 G395D; L417S; A432V + 139/140L56A; A74T; A241V; N286S; I314R; + E316W; A323T 141/142 E316H + 143/144L56A; S86A; K163F; I314R; E316W; + A383V; C414V; P416A; V422A 145/146L56A; A323T; A383V + 147/148 G395D + 149/150 E316W; A323T; D394G; C414V;P416A + 151/152 K163F; I314R; E316W; D394G + 153/154 E401A + 155/156L56C + 157/158 A74T; S86A; E316W; A323T; A383V; + D394G 159/160 A383V +161/162 E316R; + 163/164 N53C; R146H; L417S + 165/166 I314R; A383V;C414V; V422A + 167/168 A74T; E316W; A323T; D394G + 169/170 L56A; K163F +171/172 L56A; S86A; I314R; E316W; A323T; + D394G; C414V; V422A 173/174L56A; A383V + 175/176 S86A; A383V; D394G + 177/178 L56A; A323T; A383V;D394G + 179/180 D21P; N53C; L417S + 181/182 F85V; S86A; H88R; K163F;A323T; + A383V; D394G 183/184 E401S + 185/186 R146H; G395D; L417S +187/188 E316W; A323T; A383V; D394G + 189/190 E316W; C414V; V422A +191/192 G18A + 193/194 A74T; S86A; K163F; E316W + 195/196 A74T; F85V;I314R; E316W; C414V; + P416A 197/198 H88R; E316W; A323T + 199/200 P23R +201/202 S86A; H88R; K163F; A323T; A383V; + C414V; V422A 203/204 L56C;R146H; A432V + 205/206 L56T + 207/208 S86G + 209/210 A74T; F85V; S86A;K163F; N286S; + E316W; A323T; D394G 211/212 H88R; N286S; E316W; A323T +213/214 A199V; L417S + 215/216 L56A; K163F; N286S; E316W; A323T; +A383V; D394G 217/218 P23S; N53C; L417S + 219/220 S284A + 221/222 A74T;H88R; A323T; A383V + 223/224 P164D + 225/226 A149S + 227/228 C260T +229/230 K163F; E316W; A323T + 231/232 F85V; N286S + 233/234 P164S +235/236 L56V + 237/238 L56C; G395D + 239/240 A74T; N286S; D394G; P416A +241/242 I314R; A383V + 243/244 A404S + 245/246 L56A; A323T + 247/248L56A; S86A; N286S; I314R; C414V; + P416A 249/250 N53C; L56C + 251/252P23S; G395D; L417S + 253/254 S157A + 255/256 P23S; L56C + 257/258 H88T +259/260 I403V + 261/262 P23A + 263/264 L417S; A432V + 265/266 N53C +267/268 D21P; P23S; L56C; R146H + 269/270 P23S; N53C; L56C; R146H;G395D + 271/272 D21P; P23S; G395D; L417S; A432V + 273/274 D21P; N53C;L56C + 275/276 W147R; G395D; L417S; A432V + 277/278 H88S + 279/280V422L + 281/282 D21P; P23S; R146H; L417V + 283/284 E315R + 285/286 D21P;L56C; G395D + 287/288 P23S; N53C + 289/290 L417V; A432V + 291/292 L56C;W147R + 293/294 E316L + 295/296 D21P; P23S; L56C; R146H; A432V + 297/298L56A + 299/300 P23S; L56C; G395D + 301/302 E316A + 303/304 L417A +305/306 T408L + 307/308 N53C; G395D + 309/310 P23S; N53C; G395D +311/312 T20C + 313/314 P23S; N53C; A432V + 315/316 E401K + 317/318 L56C;R146H; L417V + 319/320 P23S; N53C; L56C + 321/322 L417V + 323/324R415G + 325/326 D21R + 327/328 D21P; L417S; A432V + 329/330 P164A +331/332 N405W + 333/334 W147R; L417S + 335/336 R146H; L417V + 337/338R146H + 339/340 T406S + 341/342 G395D; L417V + 343/344 N405H + 345/346R415W + 347/348 S400D + 349/350 P23S; L417V; A432V + 351/352 R146H;G395D + 353/354 S420G + 355/356 E316V + 357/358 R146H; W147R; G395D;L417S + 359/360 P23S; L56C; G395D; L417V + 361/362 P23S; L417V + ¹Levelsof increased activity were determined relative to the referencepolypeptide of SEQ ID NO: 4 and defined as follows: ″+″ 1.20 to 3.00,″++″ >3.00, ″+++″ >5.00

TABLE 2-2 ATA Activity in the Production of Compound (2) Relative to SEQID NO: 4 SEQ Percent Conversion ID Fold Improvement NO: Amino AcidDifferences (Relative to (nt/aa) (Relative to SEQ ID NO: 4) SEQ ID NO:4)¹ 7/8 K163F; N286S; I314R; E316W; A323T; +++ C414V; P416A  9/10 N286S;I314R; A323T; A383V ++ 11/12 K163F; N286S; I314R; A323T; D394G ++ 13/14N286S; E316W; A323T; A383V; ++ C414V; P416A 15/16 A74T; G81S; N286S;E316W; + A323T; A383V 5/6 T408F + 19/20 E315G + ¹Levels of increasedactivity were determined relative to the reference polypeptide of SEQ IDNO: 4 and defined as follows: ″+″ 5.00 to 7.00, ″++″ >7.00, ″+++″ >8.00

Example 3 Evolution and Screening of Engineered Polypeptides Derivedfrom SEQ ID NO: 8 for Improved Production of Compound (2)

The engineered polynucleotide (SEQ ID NO: 7) encoding the polypeptidewith transaminase activity of SEQ ID NO: 8 was used to generate theengineered polypeptides of Table 3-1. These polypeptides displayedimproved transaminase activity under the desired conditions e.g., theimprovement in the formation of the amine product, compound (2), fromthe ketone substrate, compound (1), as compared to the startingpolypeptide. The engineered polypeptides, having the amino acidsequences of even-numbered sequence identifiers were generated from the“backbone” amino acid sequence of SEQ ID NO: 8 as described below.

Directed evolution began with the polynucleotide set forth in SEQ ID NO:7. Libraries of engineered polypeptides were generated using variouswell-known techniques (e.g., saturation mutagenesis, recombination ofpreviously identified beneficial amino acid differences) and screenedusing HTP assay and analysis methods that measured the polypeptides'ability to produce compound (2).

The enzyme assay and analysis were carried out as described in Example 2except that the lysate was diluted 512-fold before adding to thereaction mixture.

Hit variants were grown in 250 mL shake flasks and enzyme powdersgenerated as described in Example 1. The activity of the enzyme powderswas evaluated at 0-32 g/L of the ATA shake flask powder, 20-200 g/Lketone, 1-2.5 M IPM (4 M pH 9 stock), 0.5 g/L PLP, 0-20% DMSO, pH˜9,40-60° C., for 20-24 hours, using similar assay as described above andthe hit variants are listed in Table 3-2.

TABLE 3-1 ATA Activity in the Production of Compound (2) Relative to SEQID NO: 8 SEQ Percent Conversion ID Fold Improvement NO: Amino AcidDifferences (Relative to (nt/aa) (Relative to SEQ ID NO: 8) SEQ ID NO:8)¹ 363/364 T408A +++ 365/366 D21H; F163L; T323C; T408F +++ 367/368F291Y +++ 369/370 R351L +++ 371/372 V272E +++ 373/374 T408E ++ 375/376A383V ++ 377/378 E365S ++ 379/380 H362Q ++ 381/382 N342T ++ 383/384 S24K++ 385/386 E189V ++ 387/388 V42F; I363L ++ 389/390 G191D ++ 391/392D439S ++ 393/394 A388L ++ 395/396 K447S ++ 397/398 V42F; V272E; F291Y ++399/400 K66A + 401/402 E365L + 403/404 A388D + 405/406 E77M + 407/408K443S + 409/410 E365R + 411/412 D107L + 413/414 G18A; P23R; A149S;A383V + 415/416 A199Q + 417/418 T277S + 419/420 G46S + 421/422 E195W +423/424 V42F; V272E; F291Y; I363L + 425/426 L417S + 427/428 V42F; V272E;I363L; L410H + 429/430 V42F; V272E; G324S; I363L; R366H + 431/432T309F + 433/434 K385L + 435/436 Y187E; F291Y + 437/438 Y187E + 439/440E189W + 441/442 K385T + 443/444 E343G + 445/446 E365Q + 447/448 Q210V +449/450 A134V + 451/452 C260T; G395D; E401S + 453/454 Q5G + 455/456A388P + 457/458 D107S + 459/460 G18A; P23R; A149S; C260T; A383V; +G395D; E401S; A416P 461/462 Q5E + 463/464 S86G; A383V + 465/466 E358L +467/468 E451S + 469/470 E189S + 471/472 E189F + 473/474 R203L + 475/476H362V + 477/478 N396P + 479/480 K443L + 481/482 K248G + 483/484 Q210A +485/486 P23R; A149S; S284A; A383V; G395D + 487/488 S167N + 489/490P138R + 491/492 G395R + 493/494 P392L + 495/496 Q210M + 497/498 A416P +499/500 S389D; + 501/502 F163L; I259V; T323C; T408F + 503/504 K361R +505/506 V42F + 507/508 D107Y + 509/510 P354S + 511/512 F163L; E315G;W316F + 513/514 P367T + 515/516 N396Y + 517/518 K447T + 519/520 Q210Y +521/522 K211R + 523/524 S24R + 525/526 I363L; R366H + 527/528 A404M +529/530 D439L + 531/532 T309A + 533/534 V42F; Y187E; A353T + 535/536V42F; V272E; L410H + 537/538 Y187E; V272E; G324S; I363L; L410H + 539/540N405W + 541/542 G191F + 543/544 K305E + 545/546 V272E; A353T + 547/548E401Q + 549/550 Q210L + 551/552 R186Q + 553/554 A450D + 555/556 T309R +557/558 P392A + 559/560 Y187E; V272E; I363L + 561/562 S86G; F163M;P164S; C260T; A383V + 563/564 P164D; W316H; A383V; E401S + 565/566 V42F;F291Y; I363L + 567/568 G18A; F163M; P164Q + 569/570 T408W + 571/572P23R; S86G + 573/574 L410H + 575/576 V272E; L410H + 577/578 R110K;Y187E + 579/580 P23R; L56C; S86G; A149S; F163M; + P164D; A383V; E401S;A416P 581/582 D21H; F163L; E315G; W316F + 583/584 F163L; I259V; T408F +585/586 A149S; A416P + 587/588 V42F; F291Y; A313V; I363L; L410H +589/590 V42F; A353T + 591/592 I259V; L307M + 593/594 I363L + 595/596P23R; A149S; C260T + 597/598 S86G; A149S; F163M; P164S; A383V; + G395D;E401S 599/600 A383V; A416P; V422T + 601/602 V42F; R110K + 603/604 V42F;F291Y; I363L; R366H + 605/606 P164S; C260T; E401S + 607/608 Y187E;V272E; I363L; R366H; L410H + 609/610 D21H + 611/612 R110K + 613/614A149S; C260T; A383V + 615/616 S86G; A149S; G395D + 617/618 V272E; I363L;R366H + 619/620 V42F; Y187E; G324S; I363L; R366H + 621/622 R110K; Y187E;V253L; L410H + 623/624 E315G + 625/626 P23R; F163M; P164Q; A383V +627/628 V42F; Y187E; V272E + 629/630 P23R; F163M; P164S; E401S; A416P +631/632 D21H; T408F + 633/634 E401S + 635/636 A383V; E401A + 637/638A149S; P164S; C260T; A383V; + G395D; E401A 639/640 Y187E; V253L; I363L;R366H + ¹Levels of increased activity were determined relative to thereference polypeptide of SEQ ID NO: 8 and defined as follows: ″+″ 1.20to 1.75, ″++″ >1.75, ″+++″ >2.00

TABLE 3-2 ATA Activity in the Production of Compound (2) Relative to SEQID NO: 8 SEQ Percent Conversion ID Fold Improvement NO: Amino AcidDifferences (Relative to (nt/aa) (Relative to SEQ ID NO: 8) SEQ ID NO:8)¹ 367/368 F291Y ++ 371/372 V272E + 365/366 D21H; F163L; T323C; T408F++ 375/376 A383V + 413/414 G18A; P23R; A149S; A383V + ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 8 and defined as follows: ″+″ 1.50 to 2.00, ″++″ >2.00

Example 4 Evolution and Screening of Engineered Polypeptides Derivedfrom SEQ ID NO: 366 for Improved Production of Compound (2)

The engineered polynucleotide (SEQ ID NO: 365) encoding the polypeptidewith transaminase activity of SEQ ID NO: 366 was used to generate theengineered polypeptides of Table 4-1. These polypeptides displayedimproved transaminase activity under the desired conditions e.g., theimprovement in the formation of the compound (2) (amine product) fromthe ketone substrate (compound (1)) as compared to the startingpolypeptide. The engineered polypeptides, having the amino acidsequences of even-numbered sequence identifiers were generated from the“backbone” amino acid sequence of SEQ ID NO: 366 as described belowtogether with the HTP assay and analytical methods described in Tables6-1 and 6-2.

The enzyme assay and analysis were carried out as described in Example 2except that the lysate was diluted 1700-fold before adding to thereaction mixture.

Hit variants were grown in 250 mL shake flasks and enzyme powdersgenerated as described in Example 1. The activity of the enzyme powderswas evaluated at 0-60 g/L of the ATA shake flask powder, 20-300 g/Lketone, 1 M IPM (4 M pH 9 stock), 0.5 g/L PLP, 0-20% DMSO, pH˜9, 40-60°C., for 20-24 hours, using similar assay as described above and the hitvariants are listed in Table 4-2.

TABLE 4-1 ATA Activity in the Production of Compound (2) Relative to SEQID NO: 366 SEQ Percent Conversion ID Fold Improvement NO: Amino AcidDifferences (Relative to (nt/aa) (Relative to SEQ ID NO: 366) SEQ ID NO:366)¹ 641/642 S24K; K66A; F291Y; E365S +++ 643/644 S24K; K66A; G191D;A199Q; +++ F291Y 645/646 G191D; F291Y; N342T; H362Q; ++ E365S 647/648F291Y; A383V ++ 649/650 L163M; F291Y; A383V; A388D ++ 651/652 S24K ++653/654 L163M; F291Y; H362Q; E365S; ++ A383V; A388D 655/656 F291Y ++657/658 V42F; F291Y; R351L; H362Q; ++ A383V; F408A 659/660 S24K; F291Y +661/662 S24K; K66A; N342T; E365S; ++ A388D; F408E 663/664 K66A; A383V ++665/666 K66A; F291Y; H362Q; E365S; ++ A383V 667/668 S24K; K66A; F291Y;N342T; ++ A383V 669/670 V42F; F291Y; A383V; A388D ++ 671/672 V42F;G191D; F408E ++ 673/674 S24K; A199Q; C260T; R351L; ++ H362Q; A383V675/676 S24K; K66A; L163M; G191D; ++ H362Q; A383V; A388D 677/678 S24K;F291Y; N342T; R351L; ++ A383V 679/680 K66A; F291Y; A383V; A388D ++681/682 A199Q; F291Y ++ 683/684 S24K; A388D ++ 685/686 A199Q; C260T;A383V ++ 687/688 H362Q ++ 689/690 L163M; A383V + 691/692 S24K; V42F;K66A; F291Y + 693/694 F291Y; R351L; A383V; A388D; + F408A 695/696 V42F;A199Q; F291Y; A383V + 697/698 S24K; A199Q; C260T; H362Q; + A383V; A388D699/700 S24K; L163M; R351L; A383V + 701/702 S24K; C260T; H362Q; A383V; +A388D 703/704 A383V + 705/706 S24K; D107L; L163M; G191D; + F291Y; R351L;A383V; A388D 707/708 F291Y; R351L; A383V; A388D + 709/710 C260T; F291Y;E365S; A383V; + F408A 711/712 G191D; C260T; A388D + 713/714 L25V +715/716 V33T + 717/718 R351L; A383V; A388D + 719/720 G191D; R351L;A383V; A388D + 721/722 N148G + 723/724 S24E + 725/726 E315S + 727/728S24K; K66A; C260T; F291Y; + A383V; A388D; F408A 729/730 L397M + 731/732N405A + 733/734 Q419S + 735/736 D107L; G191D; F291Y; A383V + 737/738L423V + 739/740 G191D; A199Q; E365S; A383V; + A388D 741/742 N342T;H362Q + 743/744 K66A; L163M; G191D; E365S; + A383V 745/746 R28S +747/748 D107L; G191D; A199Q; E365S; + A383V; A388D 749/750 S24K; E77M;F291Y + 751/752 K66A; F291Y + 753/754 S24K; A383V; A388D + 755/756H362Q; A388D + 757/758 S24K; K66A; G191D; C260T; + F408A 759/760 K66A;A199Q; R351L; A383V + 761/762 S24K; K66A; G191D; A199Q; + C260T; F291Y;R351L 763/764 S24K; G191D; F291Y; E365S + 765/766 R314K + 767/768 S24K;D107L; F291Y; R351L; + E365S; A388D 769/770 G191D; F291Y + 771/772 S24K;F291Y; F408A + 773/774 F291Y; E365S; A388D + 775/776 E77M; A383V;A388D + 777/778 L25H + 779/780 S24K; V42F; F291Y; H362Q + 781/782 F291Y;H362Q; E365S + 783/784 H319S + 785/786 V42F; F291Y; R351L; H362Q; +E365S; A383V; A388D 787/788 C260T + 789/790 C260T; E365S; A383V +791/792 E77M; F291Y + 793/794 A383V; A388D + 795/796 K66A; Y82H; F291Y;A383V + 797/798 S24K; F291Y; H362Q; A388D + 799/800 R28T + 801/802A153S + 803/804 T406H + 805/806 S86T + 807/808 W316V + 809/810 I413L +811/812 N396R + ¹Levels of increased activity were determined relativeto the reference polypeptide of SEQ ID NO: 366 and defined as follows:″+″ 1.10 to 1.50, ″++″ >1.50, ″+++″ >1.80

TABLE 4-2 ATA Activity in the Production of Compound (2) Relative to SEQID NO: 366 SEQ Percent Conversion ID Fold Improvement NO: Amino AcidDifferences (Relative to (nt/aa) (Relative to SEQ ID NO: 366) SEQ ID NO:366)¹ 645/646 G191D; F291Y; N342T; H362Q; + E365S 647/648 F291Y; A383V +649/650 L163M; F291Y; A383V; A388D + 653/654 L163M; F291Y; H362Q;E365S; + A383V; A388D 655/656 F291Y + 641/642 S24K; K66A; F291Y; E365S++ 643/644 S24K; K66A; G191D; A199Q; + F291Y ¹Levels of increasedactivity were determined relative to the reference polypeptide of SEQ IDNO: 366 and defined as follows: ″+″ 1.70 to 2.00 ″++″ >2.00

Example 5 Evolution and Screening of Engineered Polypeptides Derivedfrom SEQ ID NO: 650 for Improved Production of Compound (2)

The engineered polynucleotide (SEQ ID NO: 649) encoding the polypeptidewith transaminase activity of SEQ ID NO: 650 was used to generate theengineered polypeptides of Table 5-1. These polypeptides displayedimproved the transaminase activity under the desired conditions e.g.,the improvement in the formation of the compound (2) (amine product)from the ketone substrate (compound V) as compared to the startingpolypeptide. The engineered polypeptides, having the amino acidsequences of even-numbered sequence identifiers were generated from the“backbone” amino acid sequence of SEQ ID NO: 650 as described belowtogether with the HTP assay and analytical methods described in Tables6-1 and 6-2.

Directed evolution began with the polynucleotide set forth in SEQ ID NO:649. Libraries of engineered polypeptides were generated using variouswell-known techniques (e.g., saturation mutagenesis, recombination ofpreviously identified beneficial amino acid differences) and screenedusing HTP assay and analysis methods that measured the polypeptides'ability to production of compound (2).

The enzyme assay and analysis were carried out as described in Example 2except that the lysate was diluted 4000-fold before adding to thereaction mixture.

Hit variants were grown in 250-mL shake flask and enzyme powdersgenerated. The activity of the enzyme powders was evaluated at 0-3 g/Lof the ATA shake flask powders, 20-100 g/L ketone, 1 M IPM (4 M pH 9stock), 0.5 g/L PLP, 0-18% DMSO co-solvent, pH˜9, 50° C., for 24 hours,using similar assay as described above and the hit variants are listedin Table 5-2.

TABLE 5-1 ATA Activity in the Production of Compound (2) Relative to SEQID NO: 650 SEQ Percent Conversion ID Fold Improvement NO: Amino AcidDifferences (Relative to (nt/aa) (Relative to SEQ ID NO: 650) SEQ ID NO:650)¹ 813/814 V383L ++ 815/816 R78A ++ 817/818 W316S ++ 819/820 W316L ++821/822 W316N + 823/824 A72G + 825/826 M226Q + 827/828 Y14A + 829/830F169V + 831/832 G175D; W316F + 833/834 Y14H; S108R; A133R; I311S +835/836 T114A + 837/838 S24E; M163L; A199Q + 839/840 W316D + 841/842M95I + 843/844 T154S + 845/846 W316Y + 847/848 Y14G + 849/850 E382D +851/852 K73R + 853/854 W316V + 855/856 Y14H; I311S + 857/858 I311K +859/860 S108R + 861/862 T13A; I311S + 863/864 T13A; S24K; A199Q; I311S +865/866 T13A; S108R + 867/868 K73S + 869/870 D386A + 871/872 H35E +873/874 F169C + 875/876 V101L + 877/878 W316E + 879/880 P293A + 881/882S24E; M163L + 883/884 T13A + 885/886 R10E + 887/888 M163V + 889/890W316G + 891/892 Y14H; S24K; S108R; A199Q + 893/894 T13A; A199Q + 895/896W316H + 897/898 W316I + 899/900 W316F + 901/902 Y14H; S24K; A199Q +903/904 T13A; S24E; S108R; M163L; I311S + 905/906 Y14H + 907/908 T13A;S24E; M163L + 909/910 T13A; S24E; A133R; A199Q; I311S + 911/912 S108R;A199Q + 913/914 Y14H; S24E; S108R; A133R + 915/916 Y14H; S24K; S108R +917/918 M163H + 919/920 S24E + 921/922 Y14H; S108R + 923/924 T13A;S108R; I311S + 925/926 A199Q; I311S + 927/928 Y14H; S108R; I311S +929/930 T13A; S108R; A199Q + 931/932 A199Q + 933/934 M163S + 935/936T13A; S24K; S108R; M163L + ¹Levels of increased activity were determinedrelative to the reference polypeptide of SEQ ID NO: 650 and defined asfollows: ″+″ 1.00 to 1.35 ″++″ >1.35

TABLE 5-2 ATA Activity in the Production of Compound (2) Relative to SEQID NO: 650 SEQ Percent Conversion ID Fold Improvement NO: Amino AcidDifferences (Relative to (nt/aa) (Relative to SEQ ID NO: 650) SEQ ID NO:650)¹ 813/814 V383L ++ 815/816 R78A ++ 819/820 W316L + 817/818 W316S +821/822 W316N + 833/834 Y14H; S108R; A133R; I311S + 837/838 S24E; M163L;A199Q + 823/824 A72G + ¹Levels of increased activity were determinedrelative to the reference polypeptide of SEQ ID NO: 650 and defined asfollows: ″+″ 1.20 to 1.40 ″++″ >1.40

Example 6 Analytical Detection of Conversion of Compound (1) to Compound(2)

Data described in Examples 2 to 5 were collected using the analyticalmethods provided in Tables 6-1 and 6-2. The methods provided herein finduse in analyzing the variants produced using the present invention.However, it is not intended that present invention be limited to themethods described herein, as there are other suitable methods known inthe art that are applicable to the analysis of the variants providedherein and/or produced using the methods provided herein.

TABLE 6-1 HPLC Parameters Method for determining conversion of Compound(1) to Compound (2) Instrument Shimadzu HPLC Column Cogent DiamondHydride, 4.6 x 150 mm x 4 μm, (p/n 70000-15P) Mobile phase Isocratic:65% H₂O + 0.1% TFA, 35% MeCN + 0.1% TFA Flow rate 1.5 mL/min Run time1.6 min Peak retention Amine: 1.15 min times Ketone: 1.30 min Column 50°C. temperature Injection 5 μL volume Detection 265 nm wavelength

TABLE 6-2 HPLC Parameters Method for determining selectivity InstrumentShimadzu HPLC Column Agilent Poroshell 120 PhenylHexyl, 4.6 x 100 mm x2.7 μm (p/n 695975-912) Mobile phase A: 0.1% TFA in water, B: 0.1% TFAin MeCN Mobile phase 10% B for 2 min, step change to 100% B at 2.01 min,gradient hold at 100% B to 3 min, step change to 10% B at 3.01 min, stopat 4 min Flow rate 1.5 mL/min Run time 4.0 min Peak retention S-amine:2.4 min times R-amine: 2.6 min Ketone: 3.2 min Column 50° C. temperatureInjection 5 μL volume Detection 265 nm

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

We claim:
 1. An engineered polynucleotide encoding an engineeredtransaminase having an amino acid sequence of at least 85% identity toSEQ ID NO: 4, 8, 366, or 650, or a functional fragment thereof, and atleast one substitution or a set of substitutions in said amino acidsequence relative to SEQ ID NO: 4, wherein the positions of saidsubstitutions are numbered with reference to SEQ ID NO: 4, 8, 366, or650.
 2. The polynucleotide of claim 1, wherein the engineeredtransaminase has at least 85% sequence identity to SEQ ID NO: 4 andcomprises at least one substitution or set of substitutions at one ormore positions selected from:21/163/286/291/314/316/323/383/388/408/414/416, 18, 20, 21,21/23/56/146, 21/23/56/146/432, 21/23/146/417, 21/23/395/417/432,21/53/56, 21/53/417, 21/56/395, 21/417/432, 23, 23/53, 23/53/56,23/53/56/146/395, 23/53/395, 23/53/417, 23/53/432, 23/56, 23/56/395,23/56/395/417, 23/395/417, 23/417, 23/417/432, 53, 53/56, 53/146/417,53/395, 56, 56/74/241/286/314/316/323,56/86/163/314/316/383/414/416/422, 56/86/286/314/414/416,56/86/314/316/323/394/414/422, 56/146/417, 56/146/432, 56/147, 56/163,56/163/286/316/323/383/394, 56/286/314/316/323/422, 56/286/383, 56/323,56/323/383, 56/323/383/394, 56/383, 56/395, 74/81/286/316/323/383,74/85/86/163/286/316/323/394, 74/85/314/316/414/416, 74/86/163/316,74/86/316/323/383/394, 74/88/286/316/323/383, 74/88/323/383,74/163/286/316/383/394/416, 74/163/314/316, 74/163/314/316/323/394,74/163/314/323/383/414/416, 74/286, 74/286/316/323, 74/286/394/416,74/314/323/383/394/414, 74/316/323/394, 85/86/88/163/323/383/394,85/86/163/314/323/394/414, 85/286, 85/286/323, 86,86/88/163/323/383/414/422, 86/383/394, 88, 88/163/286/383,88/286/316/323, 88/286/316/323/383/414/416, 88/316/323, 146,146/147/395/417, 146/395, 146/395/417, 146/417, 147/395/417/432,147/417, 149, 157, 163, 163/222/286/316/323/383/394, 163/286,163/286/314/316/323/414/416, 163/286/314/323/394,163/286/316/323/394/416, 163/286/414, 163/314/316/394, 163/314/323/394,163/314/383, 163/314/414, 163/316/323, 163/323, 163/383, 164, 199/417,259, 260, 284, 286, 286/314/323/383, 286/314/394,286/316/323/383/414/416, 286/316/383/394, 286/316/394/414/416, 286/323,286/323/383/414, 286/323/416, 286/383, 286/416, 314/316, 314/316/323,314/316/323/383/422, 314/316/323/394, 314/316/394, 314/323/383/394,314/383, 314/383/414/422, 315, 316, 316/323/383/394,316/323/394/414/416, 316/414/422, 323, 323/383, 323/383/394/414/416,323/394, 383, 395, 395/417, 395/417/432, 400, 401, 403, 404, 405, 406,408, 415, 417, 417/432, 420, and
 422. 3. The polynucleotide of claim 1,wherein the engineered transaminase has at least 90% sequence identityto SEQ ID NO: 4 and comprises at least one substitution or set ofsubstitutions at one or more positions selected from:74/81/286/316/323/383, 163/286/314/316/323/414/416, 163/286/314/323/394,286/314/323/383, 286/316/323/383/414/416, 315, and 408, wherein theamino acid positions are numbered with reference to SEQ ID NO:4.
 4. Thepolynucleotide of claim 1, wherein said polypeptide sequence has atleast 85% sequence identity to SEQ ID NO: 8 and comprises at least onesubstitution or set of substitutions at one or more positions selectedfrom: 5, 18/23/149/260/383/395/401/416, 18/23/149/383, 18/163/164, 21,21/163/315/316, 21/163/323/408, 21/408,23/56/86/149/163/164/383/401/416, 23/86, 23/149/260, 23/149/284/383/395,23/163/164/383, 23/163/164/401/416, 24, 42, 42/110, 42/187/272,42/187/324/363/366, 42/187/353, 42/272/291, 42/272/291/363,42/272/324/363/366, 42/272/363/410, 42/272/410, 42/291/313/363/410,42/291/363, 42/291/363/366, 42/353, 42/363, 46, 66, 77,86/149/163/164/383/395/401, 86/149/395, 86/163/164/260/383, 86/383, 107,110, 110/187, 110/187/253/410, 134, 138, 149/164/260/383/395/401,149/260/383, 149/416, 163/259/323/408, 163/259/408, 163/315/316,164/260/401, 164/316/383/401, 167, 186, 187, 187/253/363/366,187/272/324/363/410, 187/272/363, 187/272/363/366/410, 187/291, 189,191, 195, 199, 203, 210, 211, 248, 259/307, 260/395/401, 272, 272/353,272/363/366, 272/410, 277, 291, 305, 309, 315, 342, 343, 351, 354, 358,361, 362, 363, 363/366, 365, 367, 383, 383/401, 383/416/422, 385, 388,389, 392, 395, 396, 401, 404, 405, 408, 410, 416, 417, 439, 443, 447,450, and 451, and wherein the amino acid positions are numbered withreference to SEQ ID NO:8.
 5. The polynucleotide of claim 1, wherein thepolypeptide sequence has at least 90% sequence identity to SEQ ID NO: 8,wherein said engineered transaminase comprises at least one substitutionor substitution set in said polypeptide sequence at one or morepositions selected from: 18/23/149/383, 21/163/323/408, 272, 291, and383, and wherein the amino acid positions are numbered with reference toSEQ ID NO:8.
 6. The polynucleotide of claim 1, wherein the engineeredtransaminase has at least 85% sequence identity to SEQ ID NO: 366 and atleast one substitution or set of substitutions at one or more positionsselected from: 24, 24/42/66/291, 24/42/291/362,24/66/163/191/362/383/388, 24/66/191/199/260/291/351, 24/66/191/199/291,24/66/191/260/408, 24/66/260/291/383/388/408, 24/66/291/342/383,24/66/291/365, 24/66/342/365/388/408, 24/77/291,24/107/163/191/291/351/383/388, 24/107/291/351/365/388, 24/163/351/383,24/191/291/365, 24/199/260/351/362/383, 24/199/260/362/383/388,24/260/362/383/388, 24/291, 24/291/342/351/383, 24/291/362/388,24/291/408, 24/383/388, 24/388, 25, 28, 33, 42/191/408, 42/199/291/383,42/291/351/362/365/383/388, 42/291/351/362/383/408, 42/291/383/388,66/82/291/383, 66/163/191/365/383, 66/199/351/383, 66/291,66/291/362/365/383, 66/291/383/388, 66/383, 77/291, 77/383/388, 86,107/191/199/365/383/388, 107/191/291/383, 148, 153,163/291/362/365/383/388, 163/291/383/388, 163/383, 191/199/365/383/388,191/260/388, 191/291, 191/291/342/362/365, 191/351/383/388, 199/260/383,199/291, 260, 260/291/365/383/408, 260/365/383, 291, 291/351/383/388,291/351/383/388/408, 291/362/365, 291/365/388, 291/383, 314, 315, 316,319, 342/362, 351/383/388, 362, 362/388, 383, 383/388, 396, 397, 405,406, 413, 419, and 423, and wherein the amino acid positions arenumbered with reference to SEQ ID NO:366.
 7. The polynucleotide of claim1, wherein the engineered transaminase has at least 90% sequenceidentity to SEQ ID NO: 366 and comprises at least one substitution orset of substitutions at one or more positions selected from:24/66/191/199/291, 24/66/291/365, 163/291/362/365/383/388,163/291/383/388, 191/291/342/362/365, 291, and 291/383, and wherein theamino acid positions are numbered with reference to SEQ ID NO:366. 8.The polynucleotide of claim 1, wherein the engineered transaminase hasat least 85% sequence identity to SEQ ID NO: 650 and comprises at leastone substitution or set of substitutions at one or more positionsselected from: 10, 13, 13/24/108/163, 13/24/108/163/311,13/24/133/199/311, 13/24/163, 13/24/199/311, 13/108, 13/108/199,13/108/311, 13/199, 13/311, 14, 14/24/108, 14/24/108/133, 14/24/108/199,14/24/199, 14/108, 14/108/133/311, 14/108/311, 14/311, 24, 24/163,24/163/199, 35, 72, 73, 78, 95, 101, 108, 108/199, 114, 154, 163, 169,175/316, 199, 199/311, 226, 293, 311, 316, 382, 383, and 386, andwherein the amino acid positions are numbered with reference to SEQ IDNO:650.
 9. The polynucleotide of claim 1, wherein the engineeredtransaminase has at least 90% sequence identity to SEQ ID NO: 650 andcomprises at least one substitution or set of substitutions at one ormore positions selected from: 14/108/133/311, 24/163/199, 72, 78, 316,and 383, and wherein the amino acid positions are numbered withreference to SEQ ID NO:650.
 10. The polynucleotide of claim 1, whereinsaid polynucleotide sequence is operably linked to a control sequence.11. The polynucleotide of claim 1, wherein said polynucleotide sequenceis codon optimized.
 12. The polynucleotide of claim 1, wherein theengineered transaminase exhibits an increased enzymatic activity.
 13. Anexpression vector comprising at least one polynucleotide sequence ofclaim
 1. 14. A host cell comprising at least one expression vector ofclaim
 13. 15. A host cell comprising at least one polynucleotidesequence of claim
 1. 16. A method of producing an engineeredtransaminase in a host cell, comprising culturing the host cell of claim15, under suitable conditions, such that at least one engineeredtransaminase is produced.
 17. The method of claim 16, further comprisingrecovering at least one engineered transaminase from the culture and/orhost cell.
 18. The method of claim 17, further comprising the step ofpurifying said at least one engineered transaminase.
 19. An engineeredpolypeptide having transaminase activity, wherein the polypeptidecomprises an amino acid sequence having at least 85% sequence identityto SEQ ID NO: 4, or a functional fragment thereof, wherein said aminoacid sequence comprises at least one amino acid substitution or set ofamino acid substitutions at a position or set of positions numbered withreference to SEQ ID NO: 4 selected from:21/163/286/291/314/316/323/383/388/408/414/416, 18, 20, 21,21/23/56/146, 21/23/56/146/432, 21/23/146/417, 21/23/395/417/432,21/53/56, 21/53/417, 21/56/395, 21/417/432, 23, 23/53, 23/53/56,23/53/56/146/395, 23/53/395, 23/53/417, 23/53/432, 23/56, 23/56/395,23/56/395/417, 23/395/417, 23/417, 23/417/432, 53, 53/56, 53/146/417,53/395, 56, 56/74/241/286/314/316/323,56/86/163/314/316/383/414/416/422, 56/86/286/314/414/416,56/86/314/316/323/394/414/422, 56/146/417, 56/146/432, 56/147, 56/163,56/163/286/316/323/383/394, 56/286/314/316/323/422, 56/286/383, 56/323,56/323/383, 56/323/383/394, 56/383, 56/395, 74/81/286/316/323/383,74/85/86/163/286/316/323/394, 74/85/314/316/414/416, 74/86/163/316,74/86/316/323/383/394, 74/88/286/316/323/383, 74/88/323/383,74/163/286/316/383/394/416, 74/163/314/316, 74/163/314/316/323/394,74/163/314/323/383/414/416, 74/286, 74/286/316/323, 74/286/394/416,74/314/323/383/394/414, 74/316/323/394, 85/86/88/163/323/383/394,85/86/163/314/323/394/414, 85/286, 85/286/323, 86,86/88/163/323/383/414/422, 86/383/394, 88, 88/163/286/383,88/286/316/323, 88/286/316/323/383/414/416, 88/316/323, 146,146/147/395/417, 146/395, 146/395/417, 146/417, 147/395/417/432,147/417, 149, 157, 163, 163/222/286/316/323/383/394, 163/286,163/286/314/316/323/414/416, 163/286/314/323/394,163/286/316/323/394/416, 163/286/414, 163/314/316/394, 163/314/323/394,163/314/383, 163/314/414, 163/316/323, 163/323, 163/383, 164, 199/417,259, 260, 284, 286, 286/314/323/383, 286/314/394,286/316/323/383/414/416, 286/316/383/394, 286/316/394/414/416, 286/323,286/323/383/414, 286/323/416, 286/383, 286/416, 314/316, 314/316/323,314/316/323/383/422, 314/316/323/394, 314/316/394, 314/323/383/394,314/383, 314/383/414/422, 315, 316, 316/323/383/394,316/323/394/414/416, 316/414/422, 323, 323/383, 323/383/394/414/416,323/394, 383, 395, 395/417, 395/417/432, 400, 401, 403, 404, 405, 406,408, 415, 417, 417/432, 420, and 422.